Combination therapies employing gitr binding molecules

ABSTRACT

The present invention provides combination therapies that employ a GITR binding molecule in combination with one or more additional agents.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application,60/959,246, filed on Jul. 12. 2007, titled “Combination TherapiesEmploying GITR Binding Molecules”, 61/001,021, filed on Oct. 30, 2007,titled “Combination Therapies Employing GITR Binding Molecules”, andU.S. Ser. No. 61/126,431, filed on May 5, 2008, titled “CombinationTherapies Employing GITR Binding Molecules”, the entire contents of eachare hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Cancer is one of the most prevalent health problems in the world today,affecting approximately one in five individuals in the United States. Avariety of chemotherapeutic agents are routinely employed to combatcancer. Unfortunately, many of these drugs have some toxicity at thedoses which are effective against tumors. In addition, chemotherapyresistance is a major cause of cancer treatment failure. Strategies forimproving cancer treatment have been developed over the years, but thereis still a need for effective therapies. Methods of enhancing theanti-tumor effects of chemotherapeutics would be useful for treating orreducing the advancement, severity or effects of neoplasia in subjects(e.g., humans).

SUMMARY OF THE INVENTION

The present invention is based, at least in part, on the discovery thatcombination therapies employing a GITR binding molecule, e.g., ananti-GITR antibody, and at least one additional agent, which is not aGITR binding molecule, (e.g., a chemotherapeutic agent) are moreeffective at treating and/or preventing cancer and/or reducing the sizeof certain tumors than the administration of an agent or agents withouta GITR binding molecule. Moreover, in one embodiment, a combinationtherapy of the invention has an improved safety profile. For example, inone embodiment, because the combination therapy of the invention is moreeffective, at least one of the agents may be used at a dose lower thanthat required for efficacy when used alone.

Accordingly, in one aspect the present invention provides a method forinhibiting tumor cell growth in a subject, comprising administering aGITR binding molecule, or an antigen-binding fragment thereof, and oneor more cycles of at least one additional agent to the subject, suchthat tumor cell growth is inhibited in the subject.

In another aspect, the invention provides a method for reducing tumorsize in a subject having a tumor, comprising administering a GITRbinding molecule, or an antigen-binding fragment thereof, and one ormore cycles of at least one additional agent to the subject, such thatthe tumor size is reduced.

In one embodiment, the at least one additional agent is administered tothe subject prior to administration of the GITR binding molecule, orantigen-binding fragment thereof. In another embodiment, the at leastone additional agent is administered to the subject concomitantly withthe GITR binding molecule, or antigen-binding fragment thereof. In yetanother embodiment, the at least one additional agent is administered tothe subject following administration of the GITR binding molecule, orantigen-binding fragment thereof.

In one embodiment, the at least one additional agent is achemotherapeutic agent. In one embodiment, the chemotherapeutic agent isan antimetabolite. In one embodiment, the antimetabolite is selectedfrom the group consisting of Aminopterin, Methotrexate, Pemetrexed,Raltitrexed, Cladribine, Clofarabine, Fludarabine, Mercaptopurine,Pentostatin, Thioguanine, Capecitabine, Cytarabine, Fluorouracil,Floxuridine, and Gemcitabine. In one embodiment, the antimetabolite is anucleoside analogue. In one embodiment, the nucleoside analogue isgemcitabine. In another embodiment, the nucleoside analogue isfluorouracil. In one embodiment, the chemotherapeutic agent is an agentthat affects microtubule formation. In one embodiment, the agent thataffects microtubule formation is selected from the group consisting of:paclitaxel, docetaxel, vincristine, vinblastine, vindesine, vinorelbin,taxotere, etoposide, and teniposide. In another embodiment, the agentthat affects microtubule formation is paclitaxel. In one embodiment, thechemotherapeutic agent is an alkylating agent. In one embodiment, thealkylating agent is cyclophosphamide. In one embodiment, thechemotherapeutic agent is a cytotoxic antibiotic. In one embodiment, thecytotoxic antibiotic is a topoisomerase II inhibitor. In one embodiment,the topoisomerase II inhibitor is doxorubicin.

In one embodiment, the GITR binding molecule is a humanized antibody orantibody fragment thereof. In one embodiment, the GITR binding moleculeis a human antibody or antibody fragment thereof. In one embodiment, thehumanized antibody comprises the CDRs shown in SEQ ID NOs.: 1, 2 or 3,4, 5, 6, or 7. In another embodiment, the GITR binding molecule is achimeric antibody or antibody fragment thereof.

In one embodiment, the type of tumor is selected from the groupconsisting of: pancreatic cancer, melanoma, breast cancer, lung cancer,bronchial cancer, colorectal cancer, prostate cancer, stomach cancer,ovarian cancer, urinary bladder cancer, brain or central nervous systemcancer, peripheral nervous system cancer, esophageal cancer, cervicalcancer, uterine or endometrial cancer, cancer of the oral cavity orpharynx, liver cancer, kidney cancer, testicular cancer, biliary tractcancer, small bowel or appendix cancer, salivary gland cancer, thyroidgland cancer, adrenal gland cancer, osteosarcoma, chondrosarcoma, andcancer of hematological tissues. In one embodiment, the tumor is a colontumor. In one embodiment, the colon tumor is an adenocarcinoma. Inanother embodiment, the tumor is selected from the group consisting of acolon tumor, a lung tumor, a breast tumor, a stomach tumor, a prostatetumor, a cervical tumor, a vaginal tumor, and a pancreatic tumor. In yetanother embodiment, the tumor is at a stage selected from the groupconsisting of Stage I, Stage II, Stage III, and Stage IV.

In one embodiment, the tumor is at least about 0.5 mm×0.5 mm. In anotherembodiment, the tumor is at least about 1 mm×1 mm. In yet anotherembodiment, the tumor has a volume of at least about 100 mm³.

In one embodiment, the tumor is metastatic.

In one embodiment, the administration of a GITR binding molecule, or anantigen-binding fragment thereof, and at least one chemotherapeuticagent results in. an inhibition of tumor size by at least about 42% toat least about 90%.

In another aspect, the invention provides a method for reducing tumorsize in a subject having adenocarcinoma of the colon comprisingadministering an anti-GITR antibody, or an antigen-binding fragmentthereof, and one or more cycles of gemcitabine to the subject, such thatthe tumor size is reduced.

In one embodiment, the tumor is an established tumor at the initiationof treatment.

In another aspect, the invention provides a method for reducing tumorsize in a subject having melanoma comprising administering a GITRantibody, or an antigen-binding fragment thereof, and one or more cyclesof paclitaxel to the subject, such that the tumor size is reduced.

In one embodiment, the tumor is an established tumor at the initiationof treatment. In another embodiment, the tumor is a secondary tumor atthe initiation of treatment.

In yet another aspect, the invention provides a method for reducingtumor size in a subject having adenocarcinoma of the colon comprisingadministering a GITR antibody, or an antigen-binding fragment thereof,and one or more cycles of cyclophosphamide to the subject, such that thetumor size is reduced.

In one embodiment, the tumor is an established tumor at the initiationof treatment. In another embodiment, the tumor is a secondary tumor atthe initiation of treatment.

In another aspect, the invention provides a method for reducing tumorsize in a subject having adenocarcinoma of the colon comprisingadministering a GITR antibody, or an antigen-binding fragment thereof,and one or more cycles of fluorouracil to the subject, such that thetumor size is reduced.

In one embodiment, the tumor is an established tumor at the initiationof treatment. In another embodiment, the tumor is a secondary tumor atthe initiation of treatment.

In another aspect, the invention provides a method for reducing tumorsize in a subject having adenocarcinoma of the colon comprisingadministering a GITR antibody, or an antigen-binding fragment thereof,and one or more cycles of doxorubicin to the subject, such that thetumor size is reduced.

In one embodiment, the tumor is an established tumor at the initiationof treatment. In another embodiment, the tumor is a secondary tumor atthe initiation of treatment.

In one embodiment, the anti-GITR antibody is a humanized antibody orantibody fragment thereof. In one embodiment, the humanized antibodycomprises the CDRs shown in SEQ ID NOs.:1, 2 or 3, 4, 5, 6, or 7. Inanother embodiment, the GITR binding molecule is a chimeric antibody orantibody fragment thereof.

Yet another aspect of the invention provides a kit comprising: a) apackaging material; b) a GITR binding molecule, or antigen-bindingfragment thereof; and c) a label or package insert contained within thepackaging material indicating that the GITR binding molecule, orantigen-binding fragment thereof, can be administered with at least oneadditional agent.

In one embodiment, the at least one additional agent is achemotherapeutic agent. In one embodiment, the chemotherapeutic agent isan antimetabolite. In one embodiment, the antimetabolite is a nucleosideanalogue. In one embodiment, the nucleoside inhibitor is gemcitabine. Inanother embodiment, the nucleoside analogue is fluorouracil. In oneembodiment, the chemotherapeutic agent is an agent that affectsmicrotubule formation. In one embodiment, the agent that affectsmicrotubule formation is selected from the group consisting of:paclitaxel, docetaxel, vincristine, vinblastine, vindesine, vinorelbin,taxotere, etoposide, and teniposide. In another embodiment, the agentthat affects microtubule formation is paclitaxel. In one embodiment, thechemotherapeutic agent is an alkylating agent. In one embodiment, thealkylating agent is cyclophosphamide. In one embodiment, thechemotherapeutic agent is a cytotoxic antibiotic. In one embodiment, thecytotoxic antibiotic is a topoisomerase II inhibitor. In one embodiment,the topoisomerase II inhibitor is doxorubicin.

In one embodiment, the GITR binding molecule is a humanized antibody orantibody fragment thereof. In one embodiment, the humanized antibodycomprises the CDRs shown in SEQ ID NOs.:1, 2 or 3, 4, 5, 6, or 7. Inanother embodiment, the GITR binding molecule is a chimeric antibody orantibody fragment thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts a graph showing the effect of the nucleoside analog,gemcitabine (Gemzar) (80 mg/kg), in combination with the anti-GITRantibody, 2F8 (0.4 mg), on tumor volume over the course of treatment ascompared to the effect of gemcitabine alone, 2F8 alone, and a vehiclecontrol.

FIG. 2 depicts a graph showing the effect of the nucleoside analog,gemcitabine (Gemzar) (80 mg/kg), in combination with the anti-GITRantibody, 2F8 (0.4 mg), on median survival time (Kaplan-Meier SurvivalCurve) over the course of treatment as compared to the effect ofgemcitabine alone, 2F8 alone, and a vehicle control.

FIG. 3 depicts a graph showing the effect of the nucleoside analog,gemcitabine (Gemzar) (80 mg/kg), in combination with the anti-GITRantibody, 2F8 (0.4 mg), on the number of metastatic tumors over thecourse of treatment as compared to the effect of gemcitabine alone, 2F8alone, and a vehicle control.

FIG. 4 depicts a graph showing the effect of an agent that affectsmicrotubule formation, paclitaxel (Taxol®) (10 mg/kg), in combinationwith the anti-GITR antibody, 2F8 (0.4 mg), tumor volume over the courseof treatment as compared to the effect of paclitaxel alone, 2F8 alone,and a vehicle control.

FIG. 5 depicts a graph showing the effect of the alkylating agent,cyclophosphamide (Cytoxan) (150 mg/kg), in combination with theanti-GITR antibody, 2F8 (0.4 mg), on tumor volume over the course oftreatment as compared to the effect of cyclophosphamide alone, and avehicle control.

FIG. 6 depicts a graph showing the effect of the nucleoside analog,Fluorouracil (5-FU) (75 mg/kg), in combination with the anti-GITRantibody, 2F8 (0.4 mg), on tumor volume over the course of treatment ascompared to the effect of Fluorouracil alone, and a vehicle control.

FIG. 7 depicts a graph showing the effect of the topoisomerase IIinhibitor, doxorubicin (Adriamycin) (5 mg/kg), in combination with theanti-GITR antibody, 2F8 (0.4 mg), on tumor volume over the course oftreatment as compared to the effect of Fluorouracil alone, and a vehiclecontrol.

FIG. 8 depicts a graph showing the effect of the alkylating agent,cyclophosphamide (Cytoxan) (150 mg/kg), in combination with theanti-GITR antibody, 2F8 (0.4 mg), on tumor volume over the course oftreatment as compared to the effect of cyclophosphamide alone, and avehicle control.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides, in part, methods and kits for thetreatment of cancer. More specifically, it has been shown thatcombination therapy employing an GITR binding molecule, e.g., ananti-GITR antibody, and at least one additional agent, which is not aGITR binding molecule, (e.g., a chemotherapeutic agent) is moreeffective at reducing the size of certain tumors than either agentalone.

Glucocorticoid-induced tumor necrosis factor (TNF) receptorfamily-related gene (GITR), also known as TNF receptor superfamilymember 18 (TNFRSF 18), is a type I transmembrane protein with homologyto TNF receptor family members (Nocentini G, et al. (1997) Proc NatlAcad Sci USA 94:6216-21; Gurney A L, et al. (1999) Curr Biol 9:215-8).GITR is expressed at low levels on resting CD4+ and CD8+ T cells andup-regulated following T-cell activation. Ligation of GITR provides acostimulatory signal that enhances both CD4+ and CD8+ T-cellproliferation and effector functions, (Kohm A P, et al. (2004) J Immunol172:4686-90; Kanamaru F, et al. (2004) J Immunol 172:7306-14; Ronchetti.S, et al. (2004) Eur J Immunol 34:613-22; Tone M, et al. (2003) ProcNatl Acad Sci USA 100:15059-64; Stephens G L, et al. (2004) J Immunol2004;173:5008-20). In addition, GITR is expressed constitutively at highlevels on regulatory T cells. Although GITR has previously been shown toenhance immune responses to certain protein antigens, it has notpreviously been shown to enhance the anti-tumor effects of agents usedto combat cancer.

In order that the present invention may be more readily understood,certain terms are first defined.

I. Definitions

For convenience, before further description of the present invention,certain terms employed in the specification, examples and appendedclaims are defined here.

The singular forms “a”, “an”, and “the” include plural references unlessthe context clearly dictates otherwise.

The term “administering” includes any method of delivery of apharmaceutical composition or therapeutic agent into a subject's systemor to a particular region in or on a subject. The phrases “systemicadministration,” “administered systemically”, “peripheraladministration”, and “administered peripherally” as used herein mean theadministration of a compound, drug or other material other than directlyinto the central nervous system, such that it enters the subject'ssystem and, thus, is subject to metabolism and other like processes, forexample, subcutaneous administration. “Parenteral administration” and“administered parenterally” means modes of administration other thanenteral and topical administration, usually by injection, and includes,without limitation, intravenous, intramuscular, intraarterial,intrathecal, intracapsular, intraorbital, intracardiac, intradermal,intraperitoneal, transtracheal, subcutaneous, subcuticular,intra-articular, subcapsular, subarachnoid, intraspinal and intrasternalinjection and infusion.

The term “glucocorticoid-induced TNF receptor” (abbreviated herein as“GITR”), also known as TNF receptor superfamily 18 (TNFRSF18), TEASR,and 312C2, as used herein, refers to a member of the tumor necrosisfactor/nerve growth factor receptor family. GITR is a 241 amino acidtype I transmembrane protein characterized by three cysteinepseudorepeats in the extracellular domain and specifically protectsT-cell receptor-induced apoptosis, although it does not protect cellsfrom other apoptotic signals, including Fas triggering, dexamethasonetreatment, or UV irradiation (Nocentini, G, et al. (1997) Proc. Natl.Acad. Sci., USA 94:6216-622). The nucleic acid and amino acid sequencesof human GITR (hGITR), of which there are three splice variants, areknown and can be found in, for example GenBank Accession Nos.gi:40354198, gi:23238190, gi:23238193, and gi:23238196.

The term “binding molecule” as used herein includes molecules thatcontain at least one antigen binding site that specifically binds to itstarget. For example, in one embodiment, a binding molecule for use inthe methods of the invention comprises an immunoglobulin antigen bindingsite or the portion of a ligand molecule that is responsible forreceptor binding.

In one embodiment, the binding molecule comprises at least two bindingsites. In one embodiment, the binding molecule comprises two bindingsites. In one embodiment, the binding molecules comprise three bindingsites. In another embodiment, the binding molecule comprises fourbinding sites.

The term “GITR binding molecule” refers to a molecule that comprises atleast one GITR binding site. Examples of GITR binding molecules whichare suitable for use in the methods and kits of the invention include,but are not limited to, binding molecules described in, for example,US20070098719, US20050014224, or WO05007190, each of which isincorporated in its entirety by reference herein, or binding moleculescomprising CDRs set forth in one of US20070098719, US20050014224, orWO05007190. In another embodiment, a GITR binding molecule may compriseone or more of the CDRs set forth in SEQ ID NOs.:1, 2 or 3, 4, 5, 6, or7. [SEQ ID NO.:1 (GFSLSTSGMGVG (Heavy Chain CDR1)), SEQ ID NO.:2(HIWWDDDKYYNPSLKS (HC CDR2N)), SEQ ID NO.:4 (TRRYFPFAY (HC CDR3)), SEQID NO.:5 (KASQNVGTNVA (Lignt Chain CDR1)), SEQ ID NO.:6 (SASYRYS (LCCDR2)), SEQ ID NO.:7 (QQYNTDPLT (LC CDR3)), and SEQ ID NO:3(HIWWDDDKYYQPSLKS (HC CDR2Q))]. In one embodiment, a binding moleculecomprises 1 CDR. In another embodiment, a binding molecule comprises 2CDRs. In another embodiment, a binding molecule comprises 3 CDRs. Inanother embodiment, a binding molecule comprises 4 CDRs. In anotherembodiment, a binding molecule comprises 5 CDRs. In yet anotherembodiment, a binding molecule comprises all 6 CDRs. Exemplary GITRbinding molecules suitable for use in the methods of the invention alsoinclude commercially available GITR binding molecule, such as MAB689,available from R&D Systems.

By “specifically binds” it is meant that the binding molecules exhibitessentially background binding to non-GITR molecules. An isolatedbinding molecule that specifically binds GITR may, however, havecross-reactivity to GITR molecules from other species.

As used herein, the term binding molecule includes, antibodies(including full length antibodies), monoclonal antibodies (includingfull length monoclonal antibodies), polyclonal antibodies, multispecificantibodies (e.g., bispecific antibodies), human, humanized or chimericantibodies, antibody fragments, e.g., Fab fragments, F(ab′) fragments,fragments produced by a Fab expression library, epitope-bindingfragments of any of the above, and engineered forms of antibodies (i.e.,molecules comprising binding sites derived from antibody molecules),e.g., scFv molecules or molecules comprising scFv molecule, so long asthey exhibit the desired activity, e.g., binding to GITR. In oneembodiment, the GITR binding molecules for use in the combinationtherapies of the invention bind to GITR on T cells and dendritic cells.In one embodiment, the GITR binding molecules for use in the combinationtherapies of the invention are characterized by one or more of: bindingto hGITR with high affinity, agonizing GITR activity (e.g., in thepresence of a stimulating agent, e.g., CD3), and increasing humoraland/or T cell effector responses.

In one embodiment, the binding molecules of the invention are “antibody”or “immunoglobulin” molecules, e.g., naturally occurring antibody orimmunoglobulin molecules or genetically engineered antibody moleculesthat bind antigen in a manner similar to antibody molecules. As usedherein, the term “immunoglobulin” includes a polypeptide having acombination of two heavy and two light chains whether or not itpossesses any relevant specific immunoreactivity. “Antibodies” refers tosuch assemblies which have significant known specific immunoreactiveactivity to an antigen. Antibodies and immunoglobulins comprise lightand heavy chains, with or without an interchain covalent linkage betweenthem. Basic immunoglobulin structures in vertebrate systems arerelatively well understood.

The generic term “immunoglobulin” comprises five distinct classes ofantibody that can be distinguished biochemically. All five classes ofantibodies are clearly within the scope of the present invention. Withregard to IgG, immunoglobulins comprise two identical light polypeptidechains of molecular weight approximately 23,000 Daltons, and twoidentical heavy chains of molecular weight 53,000-70,000. The fourchains are joined by disulfide bonds in a “Y” configuration wherein thelight chains bracket the heavy chains starting at the mouth of the “Y”and continuing through the variable region.

Both the light and heavy chains are divided into regions of structuraland functional homology. The terms “constant” and “variable” are usedfunctionally. In this regard, it will be appreciated that the variabledomains of both the light (VL) and heavy (VH) chain portions determineantigen recognition and specificity. Conversely, the constant domains ofthe light chain (CL) and the heavy chain (CH1, CH2 or CH3) conferimportant biological properties such as secretion, transplacentalmobility, Fc receptor binding, complement binding, and the like. Byconvention the numbering of the constant region domains increases asthey become more distal from the antigen binding site or amino-terminusof the antibody. The N-terminus is a variable region and at theC-terminus is a constant region; the CH3 and CL domains actuallycomprise the carboxy-terminus of the heavy and light chain,respectively.

Light chains are classified as either kappa or lambda (κ, λ). Each heavychain class may be bound with either a kappa or lambda light chain. Ingeneral, the light and heavy chains are covalently bonded to each other,and the “tail” portions of the two heavy chains are bonded to each otherby covalent disulfide linkages or non-covalent linkages when theimmunoglobulins are generated either by hybridomas, B cells orgenetically engineered host cells. In the heavy chain, the amino acidsequences run from an N-terminus at the forked ends of the Yconfiguration to the C-terminus at the bottom of each chain. Thoseskilled in the art will appreciate that heavy chains are classified asgamma, mu, alpha, delta, or epsilon, (γ, μ, α, δ, ε) with somesubclasses among them (e.g., γ1-γ4). It is the nature of this chain thatdetermines the “class” of the antibody as IgG, IgM, IgA IgG, or IgE,respectively. The immunoglobulin subclasses (isotypes) e.g., IgG₁, IgG₂,IgG₃, IgG₄, IgA₁, etc. are well characterized and are known to conferfunctional specialization. Modified versions of each of these classesand isotypes are readily discernable to the skilled artisan in view ofthe instant disclosure and, accordingly, are within the scope of theinstant invention.

The variable region allows the antibody to selectively recognize andspecifically bind epitopes on antigens. That is, the V_(L) domain andV_(H) domain of an antibody combine to form the variable region thatdefines a three dimensional antigen binding site. This quaternaryantibody structure forms the antigen binding site present at the end ofeach arm of the Y. More specifically, the antigen binding site isdefined by three complementary determining regions (CDRs) on each of theV_(H) and V_(L) chains.

The term “antibody”, as used herein, includes whole antibodies, e.g., ofany isotype (IgG, IgA, IgM, IgE, etc.), and includes antigen bindingfragments thereof. Exemplary antibodies include monoclonal antibodies,polyclonal antibodies, chimeric antibodies, humanized antibodies, humanantibodies, and multivalent antibodies. Antibodies may be fragmentedusing conventional techniques. Thus, the term antibody includes segmentsof proteolytically-cleaved or recombinantly-prepared portions of anantibody molecule that are capable of actively binding to a certainantigen. Non-limiting examples of proteolytic and/or recombinant antigenbinding fragments include Fab, F(ab′)2, Fab′, Fv, and single chainantibodies (sFv) containing a V[L] and/or V[H] domain joined by apeptide linker.

The binding molecules of the invention may comprise an immunoglobulinheavy chain of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY),class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule. Binding molecules may have both a heavy and alight chain.

An “antigen” is an entity (e.g., a proteinaceous entity or peptide) towhich a binding molecule specifically binds.

The term “epitope” or “antigenic determinant” refers to a site on anantigen to which a binding molecule specifically binds. Epitopes can beformed both from contiguous amino acids or noncontiguous amino acidsjuxtaposed by tertiary folding of a protein. Epitopes formed fromcontiguous amino acids are typically retained on exposure to denaturingsolvents whereas epitopes formed by tertiary folding are typically loston treatment with denaturing solvents. An epitope typically includes atleast 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 amino acids in aunique spatial conformation. Methods of determining spatial conformationof epitopes include, for example, X-ray crystallography and2-dimensional nuclear magnetic resonance. See, e.g., Epitope MappingProtocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed.(1996).

Binding molecules that recognize the same epitope can be identified in asimple immunoassay showing the ability of one antibody to block thebinding of another antibody to a target antigen, i.e., a competitivebinding assay. Competitive binding is determined in an assay in whichthe binding molecule being tested inhibits specific binding of areference binding molecule to a common antigen, such as GITR. Numeroustypes of competitive binding assays are known, for example: solid phasedirect or indirect radioimmunoassay (RIA); solid phase direct orindirect enzyme immunoassay (EIA) sandwich competition assay (see Stahliet al., Methods in Enzymology 9:242 (1983)); solid phase directbiotin-avidin EIA (see Kirkland et al., J. Immunol. 137:3614 (1986));solid phase direct labeled assay, solid phase direct labeled sandwichassay (see Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Press (1988)); solid phase direct label RIA using I-125 label(see Morel et al., Mol. Immunol. 25(1):7 (1988)); solid phase directbiotin-avidin EIA (Cheung et al., Virology 176:546 (1990)); and directlabeled RIA. (Moldenhauer et al., Scand. J. Immunol. 32:77 (1990)).Typically, such an assay involves the use of purified antigen bound to asolid surface or cells bearing either of these, an unlabeled testbinding molecule and a labeled reference binding molecule. Competitiveinhibition is measured by determining the amount of label bound to thesolid surface or cells in the presence of the test binding molecule.Usually the test binding molecule is present in excess. Usually, when acompeting binding molecule is present in excess, it will inhibitspecific binding of a reference binding molecule to a common antigen byat least 50-55%, 55-60%, 60-65%, 65-70% 70-75% or more.

An epitope is also recognized by immunologic cells, for example, B cellsand/or T cells. Cellular recognition of an epitope can be determined byin vitro assays that measure antigen-dependent proliferation, asdetermined by ³H-thymidine incorporation, by cytokine secretion, byantibody secretion, or by antigen-dependent killing (cytotoxic Tlymphocyte assay).

The term “monoclonal binding molecule” as used herein refers to abinding molecule obtained from a population of substantially homogeneousbinding molecules. Monoclonal binding molecules are highly specific,being directed against a single antigenic site. Furthermore, in contrastto polyclonal binding molecule preparations which typically includedifferent binding molecules directed against different determinants(epitopes), each monoclonal binding molecule is directed against asingle determinant on the antigen. The modifier “monoclonal” indicatesthe character of the binding molecule as being obtained from asubstantially homogeneous population of binding molecules, and is not tobe construed as requiring production of the binding molecule by anyparticular method. For example, the monoclonal binding molecules to beused in accordance with the present invention may be made by thehybridoma method first described by Kohler, et al., Nature 256:495(1975), or may be made by recombinant DNA methods (see, e.g.,U.S. Pat.No. 4,816,567). The “monoclonal binding molecules” may also be isolatedfrom phage antibody libraries using the techniques described inClackson, et al., Nature 352:624-628 (1991) and Marks et al., J. MolBiol. 222:581-597 (1991), for example.

The term “chimeric binding molecule” refers to a binding moleculecomprising amino acid sequences derived from different species. Chimericbinding molecules can be constructed, for example by geneticengineering, from binding molecule gene segments belonging to differentspecies.

The monoclonal binding molecules herein specifically include “chimeric”binding molecules in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in bindingmolecules derived from a particular species or belonging to a particularantibody class or subclass, while the remainder of the chain(s) isidentical with or homologous to corresponding sequences in bindingmolecules derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such binding molecules, solong as they exhibit the desired biological activity (U.S. Pat. No.4,816,567; and Morrison, et al., Proc. Natl. Acad. Sci. USA 81:6851-6855(1984)) e.g., binding to GITR, e.g., human GITR (hGITR) and increasing Teffector and/or humoral responses.

“Humanized” forms of non-human (e.g., murine) binding molecules areantibodies which contain minimal sequence derived from non-human bindingmolecule. For the most part, humanized binding molecules are humanbinding molecules (acceptor/recipient binding molecule) in which the CDRresidues from the hyper-variable region are replaced by CDR residuesfrom a hypervariable region of a non-human species (donor bindingmolecule) such as mouse, rat, rabbit or nonhuman primate having thedesired specificity, affinity, and capacity. In some instances, Fvframework region (FR) residues of the human binding molecule arealtered, e.g., replaced by or substituted with non-donor residues (e.g.,germline residues), or backmutated to corresponding donor humanresidues. Furthermore, humanized binding molecules may comprise residueswhich are not found in the recipient binding molecule. or in the donorbinding molecule. These modifications are generally made to furtherrefine binding molecule performance. In general, the humanized bindingmolecule will comprise substantially all of at least one, and typicallytwo, variable domains, in which all or substantially all of thehypervariable loops correspond to those of a non-human binding moleculeand all or substantially all of the FR regions are those of a humanbinding molecule sequence. The humanized binding molecule optionallyalso will comprise at least a portion of a binding molecule constantregion (Fc), typically that of a human binding molecule. For furtherdetails, see Jones, et al., Nature 321:522-525 (1986); Riechmann, etal., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.2:593-596 (1992).

The term “multispecific” includes binding molecules having specificityfor more than one target antigen. Such molecules have more than onebinding site where each binding site specifically binds (e.g.,immunoreacts with) a different target molecule or a different antigenicsite on the same target.

In one embodiment, a multispecific binding molecule of the invention isa bispecific molecule (e.g., antibody, minibody, domain deletedantibody, or fusion protein) having binding specificity for at least twotargets, e.g., more than one target molecule or more than one epitope onthe same target molecule.

In one embodiment, modified forms of antibodies can be made from a wholeprecursor or parent antibody using techniques known in the art.Exemplary techniques are discussed in more detail below. In particularlypreferred embodiments both the variable and constant regions ofpolypeptides of the invention are human. In one embodiment, fully humanantibodies can be made using techniques that are known in the art. Forexample, fully human antibodies against a specific antigen can beprepared by administering the antigen to a transgenic animal which hasbeen modified to produce such antibodies in response to antigenicchallenge, but whose endogenous loci have been disabled. Exemplarytechniques that can be used to make antibodies are described in U.S.Pat. Nos. 6,150,584; 6,458,592; 6,420,140. Other techniques, such as theuse of libraries, are known in the art.

In one embodiment, a binding molecule of the invention comprises anantibody molecule, e.g., an intact antibody molecule, or a fragment ofan antibody molecule. In another embodiment, a binding molecule of theinvention is a modified or synthetic antibody molecule. In oneembodiment, a binding molecule of the invention comprises all or aportion of (e.g., at least one antigen binding site from, at least oneCDR from) a monoclonal antibody, a humanized antibody, a chimericantibody, or a recombinantly produced antibody.

In embodiments where the binding molecule is an antibody or modifiedantibody, the antigen binding site and the heavy chain portions need notbe derived from the same immunoglobulin molecule. In this regard, thevariable region may be derived from any type of animal that can beinduced to mount a humoral response and generate immunoglobulins againstthe desired antigen. As such, the variable region of the polypeptidesmay be, for example, of mammalian origin e.g., may be human, murine,rat, non-human primate (such as cynomolgus monkeys, macaques, etc.),lupine, camelid (e.g., from camels, llamas and related species). Inanother embodiment, the variable region may be condricthoid in origin(e.g., from sharks).

In one embodiment, the binding molecules of the invention are modifiedantibodies. As used herein, the term“engineered” or “modified antibody”includes synthetic forms of antibodies which are altered such that theyare not naturally occurring, e.g., antibodies that do not comprisecomplete heavy chains (such as, domain deleted antibodies orminibodies); multispecific forms of antibodies (e.g., bispecific,trispecific, etc.) altered to bind to two or more different antigens orto different epitopes on a single antigen); heavy chain molecules joinedto scFv molecules and the like. ScFv molecules are known in the art andare described, e.g., in U.S. Pat. No. 5,892,019. In addition, the term“engineered” or “modified antibody” includes multivalent forms ofantibodies (e.g., trivalent, tetravalent, etc., antibodies that bind tothree or more copies of the same antigen or different antigens ordifferent epitopes on the same antigen).

In one embodiment, the term, “modified antibody” according to thepresent invention includes immunoglobulins, antibodies, orimmunoreactive fragments or recombinant forms thereof, in which at leasta fraction of one or more of the constant region domains has beendeleted or otherwise altered (e.g., mutated) so as to provide desiredbiochemical characteristics such as the ability to non-covalentlydimerize, increased ability to localize at the site of a tumor, oraltered serum half-life when compared with a whole, unaltered antibodyof approximately the same immunogenicity.

In one embodiment, the binding molecules of the invention may bemodified to reduce their immunogenicity using art-recognized techniques.For example, antibodies or polypeptides of the invention can behumanized, deimmunized, or chimeric antibodies can be made. These typesof antibodies are derived from a non-human antibody, typically a murineantibody, that retains or substantially retains the antigen-bindingproperties of the parent antibody, but which is less immunogenic inhumans. This may be achieved by various methods, including (a) graftingthe entire non-human variable domains onto human constant regions togenerate chimeric antibodies; (b) grafting at least a part of one ormore of the non-human complementarity determining regions (CDRs) into ahuman framework and constant regions with or without retention ofcritical framework residues; or (c) transplanting the entire non-humanvariable domains, but “cloaking” them with a human-like section byreplacement of surface residues. Such methods are disclosed in Morrisonet al., Proc. Natl. Acad. Sci. 81: 6851-5 (1984); Morrison et al., Adv.Immunol. 44: 65-92 (1988); Verhoeyen et al., Science 239: 1534-1536(1988); Padlan, Molec. Immun. 28: 489-498 (1991); Padlan, Molec. Immun.31: 169-217 (1994), and U.S. Pat. Nos. 5,585,089, 5,693,761 and5,693,762 all of which are hereby incorporated by reference in theirentirety.

The term “chemotherapeutic agent”, used interchangeably herein with“chemotherapy agent” and “antineoplastic agent”, refers to a substancethat inhibits or prevents the viability and/or function of cells, and/orcauses destruction of cells (cell death), and/or exertsanti-neoplastic/anti-proliferative effects, for example, preventsdirectly or indirectly the development, maturation or spread ofneoplastic tumor cells. The term also includes such agents that cause acytostatic effect only and not a mere cytotoxic effect. As used hereinthe term chemotherapeutic agents includes anti-angiogenic agents,tyrosine kinase inhibitors, protein kinase A inhibitors, members of thecytokine family, and radioactive isotopes.

Suitable chemotherapeutic agents according to the invention arepreferably natural or synthetic chemical compounds. There are largenumbers of anti-neoplastic chemical agents available in commercial use,in clinical evaluation and in pre-clinical development, which may beused in the combination therapies of the invention (discussed below).

The term “biologic” or “biologic agent” refers to any pharmaceuticallyactive agent made from living organisms and/or their products which isintended for use as a therapeutic, e.g., toxins such as enzymaticallyactive toxins of bacterial, fungal, plant or animal origin. In oneembodiment of the invention, biologic agents which can be used incombination with a GITR binding molecule include, but are not limited toe.g., antibodies, nucleic acid molecules, e.g., antisense nucleic acidmolecules, polypeptides or proteins. Such biologics can be administeredin combination with a GITR binding molecule by administration of thebiologic agent, e.g., prior to the administration of the GITR bindingmolecule, concomitantly with the GITR binding molecule, or after theGITR binding molecule.

The term “combination therapy”, as used herein, refers to a therapeuticregimen comprising, e.g., a GITR binding molecule and at least oneadditional non-GITR binding molecule, e.g., a chemotherapeutic agent.The GITR binding molecule and the at least one additional agent may beformulated for separate administration or may be formulated foradministration together. In one embodiment, the at least one additionalagent is not a molecule to which an immune response is desired, e.g., isnot a vaccine.

The term “cancer” or “neoplasia” refers in general to a malignantneoplasm or spontaneous growth or proliferation of cells. Cancer cellsare often in the form of a tumor, but such cells may exist alone withina subject, or may be non-tumorigenic cancer cells, such as leukemiacells. As used herein, the term “cancer” includes pre-malignant as wellas malignant cancers.

A subject having “cancer”, for example, may have a leukemia, lymphoma,or other malignancy of blood cells. In one embodiment, cancer isselected from the group consisting of pancreatic cancer, melanoma andother forms of skin cancer (e.g., squamous cell carcinoma) breastcancer, lung cancer, bronchial cancer, colorectal cancer, prostatecancer, stomach cancer, ovarian cancer, brain or central nervous systemcancer, peripheral nervous system cancer, esophageal cancer, cervicalcancer, uterine or endometrial cancer, cancer of the head and neck(including cancer of the oral cavity or nasopharynx), liver and biliarytract cancer, kidney and renal collecting system, including urinarybladder cancer, testicular cancer, small bowel or appendix cancer,salivary gland cancer, thyroid gland cancer, adrenal gland cancer,sarcomas (including osteosarcoma and chondrosarcoma), and cancer ofhematological tissues.

In certain embodiments, the subject methods are used to treat a solidtumor. Exemplary solid tumors include but are not limited to small andnon-small cell lung cancer (NSCLC), testicular cancer, ovarian cancer,uterine cancer, cervical cancer, pancreatic cancer, colorectal cancer(CRC), breast cancer, as well as prostate, gastric, skin, stomach,esophageal, and bladder cancer.

In one embodiment, a solid tumor is an adenocarcinoma, e.g., of thecolon. In one embodiment of the invention, a solid tumor is a colontumor. In another embodiment of the invention, a solid tumor is selectedfrom the group consisting of a colon tumor, a lung tumor, a breasttumor, a stomach tumor, a prostate tumor, a cervical tumor, a vaginaltumor, and a pancreatic tumor.

In one embodiment of the invention, the cancer to be treated is amelanoma.

In certain embodiments of the invention, the subject methods are used toreduce and/or prevent tumor cell proliferation. In certain embodimentsof the invention, the subject methods are used to reduce and/or preventtumor metastasis. In another embodiment, the subject methods are used toreduce the size of a tumor, e.g., an established tumor, and/or asecondary tumor, e.g., a metastasis. As used herein, an “establishedtumor” is a solid tumor of sufficient size such that nutrients, i.e.,oxygen can no longer permeate to the center of the tumor from thesubject's vasculature by osmosis and, therefore, the tumor requires itsown vascular supply to receive nutrients.

In one embodiment, the subject methods are used to treat a vascularizedtumor. The term “vascularized tumor” includes tumors having thehallmarks of established vasculature. Such tumors are identified bytheir size and/or by the presence of markers associated with bloodvessels or angiogenesis. In one embodiment, the tumor is at least about0.5 mm×0.5 mm. In another embodiment, the tumor is at least about 1 mm×1mm. In yet another embodiment, the tumor has a volume of at least about100 mm³. In another embodiment, the tumor has a volume of at least about200 mm³. In another embodiment, the tumor has a volume of at least about300 mm³. In another embodiment, the tumor has a volume of at least about400 mm³. In another embodiment, the tumor has a volume of at least about500 mm³. In one embodiment, the tumor is large enough to be found bypalpation or by using art recognized imaging techniques.

In another embodiment, the subject methods are used to treat a solidtumor that is not quiescent and is actively undergoing exponentialgrowth. In another embodiment, the subject methods are used to treat asmall tumor, such as a micrometastasis, e.g., a tumor detectable only byhistological examination but not by other techniques.

The term “effective amount” refers to that amount of combination therapywhich is sufficient to produce a desired result on a cancerous cell ortumor, including, but not limited to, for example, reducing tumor sizeand/or reducing tumor volume of a solid tumor, either in vitro or invivo. In one embodiment of the invention, an effective amount of acombination therapy is the amount that results in an inhibition of tumorsize more than about 10%, more than about 20%, more than about 30%, morethan about 35%, more than about 42%, more than about 43%, more thanabout 44%, more than about 45%, more than about 46%, more than about47%, more than about 48%, more than about 49%, more than about 50%, morethan about 51%, more than about 52%, more than about 53%, more thanabout 54%, more than about 55%, more than about 56%, more than about57%, more than about 58%, more than about 59%, more than about 60%, morethan about 65%, more than about 70%, more than about 75%, more thanabout 80%, more than about 85%, more than about 90%, more than about95%, or more than about 100%.

The term also includes that amount of a combination therapy which issufficient to achieve a desired clinical result, including but notlimited to, for example, preventing recurrence, ameliorating disease,stabilizing a patient, preventing or delaying the development ofmetastasis, or preventing or slowing the progression of cancer in apatient. An effective amount of the combination therapy can bedetermined based on one administration of each of the agents or repeatedadministration of at lest one of the agents of the therapy. Methods ofdetection and measurement of the indicators above are known to those ofordinary skill in the art. Such methods include, but are not limited tomeasuring reduction in tumor burden, reduction of tumor size, reductionof tumor volume, reduction in proliferation of secondary tumors,decreased solid tumor vascularization, alteration in the expression ofgenes in tumor tissue or adjacent tissue, presence or absence ofbiomarkers, lymph node involvement, histologic grade, detecting the lackof recurrence of a tumor, a reduced rate of tumor growth, reduced tumorcell metabolism, and/or nuclear grade.

In one embodiment of the invention, tumor burden is determined. “Tumorburden” also referred to as “tumor load”, refers to the total amount oftumor material distributed throughout the body. Tumor burden refers tothe total number of cancer cells or the total size of tumor(s),throughout the body, including lymph nodes and bone barrow. Tumor burdencan be determined by a variety of methods known in the art, such as,e.g. by measuring the dimensions of tumor(s) upon removal from thesubject, e.g., using calipers, or while in the body using imagingtechniques, e.g., ultrasound, bone scan, computed tomography (CT) ormagnetic resonance imaging (MRI) scans.

In one embodiment of the invention, tumor size is determined. The term“tumor size” refers to the total size of the tumor which can be measuredas the length and width of a tumor. Tumor size may be determined by avariety of methods known in the art, such as, e.g. by measuring thedimensions of tumor(s) upon removal from the subject, e.g., usingcalipers, or while in the body using imaging techniques, e.g., bonescan, ultrasound, CT or MRI scans.

In one embodiment of the invention, tumor size is determined bydetermining tumor weight. In one embodiment, tumor weight is determinedby measuring the length of the tumor, multiplying it by the square ofthe width of the tumor, and dividing that sum by 2.

In one embodiment of the invention, tumor size is determined bydetermining tumor volume. The term “tumor volume” refers to the totalsize of the tumor, which includes the tumor itself plus affected lymphnodes if applicable. Tumor volume may be determined by a variety ofmethods known in the art, such as, e.g. by measuring the dimensions oftumor(s) upon removal from the subject, e.g., using calipers, or whilein the body using an imaging techniques, e.g., ultrasound, CT or MRIscans, and calculating the volume using equations based on, for example,the z-axis diameter, or on standard shapes such as the sphere,ellipsoid, or cube. In one embodiment, tumor volume (mm³) is calculatedfor a prolate ellipsoid from 2-dimensional tumor measurements: tumorvolume (mm³)=(length×width² [L×W²])÷2. Assuming unit density, tumorvolume is converted to tumor weight (i.e., 1 mm³=1 mg).

The term “vascularization of a solid tumor” refers to the formation ofblood vessels in a solid tumor. Tumor vacularization may be determinedby a variety of methods known in the art, such as, e.g. byimmunohistochemical analysis of biopsy specimens, or by imagingtechniques, such as sonography of the tumor, angiography, CT or magneticMRI scans.

The term “% T/C” is the percentage of the mean tumor weight of theTreatment group (T) divided by the mean tumor weight of the Controlgroup (C) multiplied by 100. A % T/C value of 42% or less is consideredindicative of meaningful activity by the National Cancer Institute(USA).

The term “% inhibition” with respect to T/C is calculated by subtractingthe % T/C from 100.

The term “statistically significant” or “statistical significance”refers to the likelihood that a result would have occurred by chance,given that an independent variable has no effect, or, that a presumednull hypothesis is true. Statistical significance can be determined byobtaining a “P-value” (P) which refers to the probability value. Thep-value indicates how likely it is that the result obtained by theexperiment is due to chance alone. In one embodiment of the invention,statistical significance can be determined by obtaining the p-value ofthe Two-Tailed One-Sample T-Test. A p-value of less than 0.05 isconsidered statistically significant, that is, not likely to be due tochance alone. Alternatively a statistically significant p-value may bebetween about 0.05 to about 0.04; between about 0.04 to about 0.03;between about 0.03 to about 0.02; between about 0.02 to about 0.01.Ranges intermediate to the above recited values, e.g., are also intendedto be part of this invention. In certain cases, the p-value may be lessthan 0.01. The p-value may be used to determine whether or not there isany statistically significant reduction in tumor size and/or anystatistically significant increase in survival when combination therapyis used to treat a subject having a tumor.

“Treating cancer” or “treating a subject having cancer” includesinhibition of the replication of cancer cells, inhibition of the spreadof cancer, reduction in tumor size, lessening or reducing the number ofcancerous cells in the body, and/or amelioration or alleviation of thesymptoms of cancer. A treatment is considered therapeutic if there is adecrease in mortality and/or morbidity, and may be performedprophylactically, or therapeutically.

A “patient” or “subject” or “host” refers to either a human being ornon-human animal.

Various aspects of the invention are described in further detail in thefollowing subsections.

II. GITR Binding Molecules

GITR binding molecules for use in the methods of the invention includebinding molecules that specifically bind to GITR and act as a GITRagonist (as demonstrated by, e.g., increased effector T cell responseand/or increased humoral immunity), such as, for example, those bindingmolecules described in US20070098719, US20050014224, and WO05007190.

In one embodiment, the GITR binding molecule is an anti-GITR antibody.Various forms of anti-GITR antibodies can be made using standardrecombinant DNA techniques (Winter and Milstein, Nature, 349, pp. 293-99(1991)).

In certain embodiments, the GITR binding molecule may be a polyclonalantibody. For example, antibodies may be raised in mammals by multiplesubcutaneous or intraperitoneal injections of the relevant antigen andan adjuvant. This immunization typically elicits an immune response thatcomprises production of antigen-reactive antibodies from activatedsplenocytes or lymphocytes. The resulting antibodies may be harvestedfrom the serum of the animal to provide polyclonal preparations.

Chimeric and/or humanized binding molecules (i.e., chimeric and/orhumanized immunoglobulins) specific for GITR are also suitable for usein the methods of the invention. Chimeric and/or humanized bindingmolecules have the same or similar binding specificity and affinity as amouse or other nonhuman binding molecules that provide the startingmaterial for construction of a chimeric or humanized binding molecule.

A chimeric binding molecule is one whose light and heavy chain geneshave been constructed, typically by genetic engineering, fromimmunoglobulin gene segments belonging to different species. Forexample, the variable (V) segments of the genes from a mouse monoclonalbinding molecule may be joined to human constant (C) segments, such asIgG1 or IgG4. Human isotype IgG1 is preferred. An exemplary chimericbinding molecule is thus a hybrid protein consisting of the V orantigen-binding domain from a mouse binding molecule and the C oreffector domain from a human binding molecule.

In one embodiment, a binding molecule suitable for use in the methods ofthe invention comprises a humanized variable region of the 6C8 bindingmolecule. In one embodiment, a binding molecule of the inventioncomprises at least one humanized 6C8 binding molecule variable region,e.g., a light chain or heavy chain variable region.

As set forth above, the term “humanized binding molecule” refers to abinding molecule comprising at least one chain comprising variableregion framework residues derived from a human binding molecule chain(referred to as the acceptor antibody or binding molecule) and at leastone complementarity determining region derived from a mouse-bindingmolecule, (referred to as the donor antibody or binding molecule).Humanized binding molecules can be produced using recombinant DNAtechnology. See for example, e.g., Hwang, W. Y. K., et al. (2005)Methods 36:35; Queen et al., Proc. Natl. Acad. Sci. USA, (1989),86:10029-10033; Jones et al., Nature, (1986), 321:522-25; Riechmann etal., Nature, (1988), 332:323-27; Verhoeyen et al., Science, (1988),239:1534-36; Orlandi et al., Proc. Natl. Acad. Sci. USA, (1989),86:3833-37; U.S. Pat. Nos. 5,225,539; 5,530,101; 5,585,089; 5,693,761;5,693,762; 6,180,370, Selick et al., WO 90/07861, and Winter, U.S. Pat.No. 5,225,539 (incorporated by reference in their entirety for allpurposes). The constant region(s), if present, are preferably alsoderived from a human immunoglobulin.

In certain embodiments, the humanized antibody is humanized 6C8 orantibody fragment thereof, as described, including the nucleotide andamino acid sequence thereof, in US20070098719. In one embodiment, thehumanized antibody comprises one or more of the CDRs shown in SEQ IDNOs.:1, 2 or 3, 4, 5, 6, or 7. In one embodiment, the humanized antibodycomprises CDRs 1, 2 or 3, 4, 5, 6, and 7.

The humanized binding molecules preferably exhibit a specific bindingaffinity for antigen of at least 10⁷, 10⁸, 10⁹, 10¹⁰, 10¹¹, or 10¹²M⁻¹.Usually the upper limit of binding affinity of the humanized bindingmolecules for antigen is within a factor of three, four or five of thatof the donor immunoglobulin. Often the lower limit of binding affinityis also within a factor of three, four or five of that of donorimmunoglobulin. Alternatively, the binding affinity can be compared tothat of a humanized binding molecule having no substitutions (e.g., abinding molecule having donor CDRs and acceptor FRs, but no FRsubstitutions). In such instances, the binding of the optimized bindingmolecule (with substitutions) is preferably at least two- to three-foldgreater, or three- to four-fold greater, than that of the unsubstitutedbinding molecule. For making comparisons, activity of the variousbinding molecules can be determined, for example, by BIACORE (i.e.,surface plasmon resonance using unlabelled reagents) or competitivebinding assays.

In certain embodiments, a GITR binding molecule is a chimeric antibody.In one embodiment, a chimeric antibody of the invention may be achimeric 6C8 antibody which is described in U.S. Patent Publication No.US20070098719, the contents of which are expressly incorporated hereinby reference.

In certain embodiments, a GITR binding molecule is a monoclonalantibody. In one embodiment, a monoclonal antibody of the invention maybe a humanized 6C8 antibody which is also described in U.S. PatentPublication No. US20070098719.

In another embodiment, a binding molecule of the invention comprises atleast one CDR derived from a murine human GITR binding molecule, e.g., a6C8 binding molecule. In another embodiment, a binding molecule of theinvention comprises at least one CDR (e.g., 1, 2, 3, 4, 5, or 6 CDRs)derived from a rat GITR binding molecule, e.g., a 2F8 binding molecule.As used herein the term “derived from” a designated protein refers tothe origin of the polypeptide. In one embodiment, the polypeptide oramino acid sequence which is derived from a particular startingpolypeptide is a CDR sequence or sequence related thereto. In anotherembodiment, the polypeptide or amino acid sequence which is derived froma particular starting polypeptide is a framework (FR) sequence orsequence related thereto. In one embodiment, the amino acid sequencewhich is derived from a particular starting polypeptide is notcontiguous.

For example, in one embodiment, one, two, three, four, five, or six CDRsare derived from a murine 6C8 antibody. In one embodiment, a bindingmolecule of the invention comprises at least one heavy or light chainCDR of a murine 6C8 antibody. In another embodiment, a binding moleculeof the invention comprises at least two CDRs from a murine 6C8 antibody.In another embodiment, a binding molecule of the invention comprises atleast three CDRs from a murine 6C8 antibody. In another embodiment, abinding molecule of the invention comprises at least four CDRs from amurine 6C8 antibody. In another embodiment, a binding molecule of theinvention comprises at least five CDRs from a murine 6C8 antibody. Inanother embodiment, a binding molecule of the invention comprises atleast six CDRs from a murine 6C8 antibody.

In one embodiment, a binding molecule of the invention comprises apolypeptide or amino acid sequence that is essentially identical to thatof a 6C8 antibody, or a portion thereof, e.g., a CDR, wherein theportion consists of at least 3-5 amino acids, of at least 5-10 aminoacids, at least 10-20 amino acids, at least 20-30 amino acids, or atleast 30-50 amino acids, or which is otherwise identifiable to one ofordinary skill in the art as having its origin in the starting sequence.

In another embodiment, the polypeptide or amino acid sequence which isderived from a particular starting polypeptide or amino acid sequenceshares an amino acid sequence identity that is about 80%, 85%, 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, with a 6C8antibody or portion thereof (e.g., a CDR) or which is otherwiseidentifiable to one of ordinary skill in the art as having its origin inthe starting sequence.

It will also be understood by one of ordinary skill in the art that ananti-GITR binding molecule for use in the methods of the invention maybe modified such that it varies in amino acid sequence from the moleculefrom which it was derived. For example, nucleotide or amino acidsubstitutions leading to conservative substitutions or changes at“non-essential” amino acid residues may be made (e.g., in CDR and/orframework residues) and maintain, increase, or decrease the ability tobind to GITR, e.g., human GITR.

An isolated nucleic acid molecule encoding a non-natural variant of apolypeptide can be created by introducing one or more nucleotidesubstitutions, additions or deletions into the nucleotide sequence ofthe binding molecule such that one or more amino acid substitutions,additions or deletions are introduced into the encoded protein.Mutations may be introduced by standard techniques, such assite-directed mutagenesis and PCR-mediated mutagenesis. In oneembodiment, conservative amino acid substitutions are made at one ormore non-essential amino acid residues. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined in the art,including basic side chains (e.g., lysine, arginine, histidine), acidicside chains (e.g., aspartic acid, glutamic acid), uncharged polar sidechains (e.g., glycine, asparagine, glutamine, serine, threonine,tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine,leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine) andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine). Thus, a nonessential amino acid residue in a bindingmolecule polypeptide may be replaced with another amino acid residuefrom the same side chain family. In another embodiment, a string ofamino acids can be replaced with a structurally similar string thatdiffers in order and/or composition of side chain family members.

Alternatively, in another embodiment, mutations may be introducedrandomly along all or part of the binding molecule coding sequence.

Preferred binding molecules for use in the methods of the inventioncomprise framework and constant region amino acid sequences derived froma human amino acid sequence. However, binding molecules may compriseframework and/or constant region sequences derived from anothermammalian species. For example, a primate framework region (e.g.,non-human primate), heavy chain portion, and/or hinge portion may beincluded in the subject binding molecules. In one embodiment, one ormore murine amino acids may be present in the framework region of abinding polypeptide, e.g., a human or non-human primate framework aminoacid sequence may comprise one or more amino acid substitutions and/orbackmutations in which the corresponding murine amino acid residue ispresent. Preferred binding molecules of the invention are lessimmunogenic than the starting 6C8 murine antibody.

The preparation of monoclonal antibodies is a well-known process (Kohleret al., Nature, 256:495 (1975)) in which the relatively short-lived, ormortal, lymphocytes from a mammal which has been injected with antigenare fused with an immortal tumor cell line (e.g. a myeloma cell line),thus, producing hybrid cells or “hybridomas” which are both immortal andcapable of producing the genetically coded antibody of the B cell. Theresulting hybrids are segregated into single genetic strains byselection, dilution, and regrowth with each individual strain comprisingspecific genes for the formation of a single antibody. They produceantibodies which are homogeneous against a desired antigen and, inreference to their pure genetic parentage, are termed “monoclonal.”

Hybridoma cells thus prepared are seeded and grown in a suitable culturemedium that preferably contains one or more substances that inhibit thegrowth or survival of the unfused, parental myeloma cells. Those skilledin the art will appreciate that reagents, cell lines and media for theformation, selection and growth of hybridomas are commercially availablefrom a number of sources and standardized protocols are wellestablished. Generally, culture medium in which the hybridoma cells aregrowing is assayed for production of monoclonal antibodies against thedesired antigen. Preferably, the binding specificity of the monoclonalantibodies produced by hybridoma cells is determined byimmunoprecipitation or by an in vitro assay, such as a radioimmunoassay(RIA) or enzyme-linked immunoabsorbent assay (ELISA). After hybridomacells are identified that produce antibodies of the desired specificity,affinity and/or activity, the clones may be subcloned by limitingdilution procedures and grown by standard methods (Goding, MonoclonalAntibodies: Principles and Practice, pp 59-103 (Academic Press, 1986)).It will further be appreciated that the monoclonal antibodies secretedby the subclones may be separated from culture medium, ascites fluid orserum by conventional purification procedures such as, for example,protein-A, hydroxylapatite chromatography, gel electrophoresis, dialysisor affinity.

In another embodiment, DNA encoding a desired monoclonal antibody may bereadily isolated and sequenced using conventional procedures (e.g., byusing oligonucleotide probes that are capable of binding specifically togenes encoding the heavy and light chains of murine antibodies). Theisolated and subcloned hybridoma cells serve as a preferred source ofsuch DNA. Once isolated, the DNA may be placed into expression vectors,which are then transfected into prokaryotic or eukaryotic host cellssuch as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO)cells or myeloma cells that do not otherwise produce immunoglobulins.More particularly, the isolated DNA (which may be modified as describedherein) may be used to clone constant and variable region sequences forthe manufacture antibodies as described in Newman et al., U.S. Pat. No.5,658,570, filed Jan. 25, 1995, which is incorporated by referenceherein. Essentially, this entails extraction of RNA from the selectedcells, conversion to cDNA, and amplification by PCR using Ig specificprimers. Suitable primers for this purpose are also described in U.S.Pat. No. 5,658,570. As will be discussed in more detail below,transformed cells expressing the desired antibody may be grown up inrelatively large quantities to provide clinical and commercial suppliesof the immunoglobulin.

Those skilled in the art will also appreciate that DNA encodingantibodies or antibody fragments may also be derived from antibody phagelibraries, e.g., using pd phage or Fd phagemid technology. Exemplarymethods are set forth, for example, in EP 368 684 B1; U.S. Pat. No.5,969,108, Hoogenboom, H. R. and Chames. 2000. Immunol. Today 21:371;Nagy et al. 2002. Nat. Med. 8:801; Huie et al. 2001. Proc. Natl. Acad.Sci. USA 98:2682; Lui et al. 2002. J. Mol. Biol. 315:1063, each of whichis incorporated herein by reference. Several publications (e.g., Markset al. Bio/Technology 10:779-783 (1992)) have described the productionof high affinity human antibodies by chain shuffling, as well ascombinatorial infection and in vivo recombination as a strategy forconstructing large phage libraries. In another embodiment, Ribosomaldisplay can be used to replace bacteriophage as the display platform(see, e.g., Hanes et al. 2000. Nat. Biotechnol. 18:1287; Wilson et al.2001. Proc. Natl. Acad. Sci. USA 98:3750; or Irving et al. 2001 J.Immunol. Methods 248:31. In yet another embodiment, cell surfacelibraries can be screened for antibodies (Boder et al. 2000. Proc. Natl.Acad. Sci. USA 97:10701; Daugherty et al. 2000 J. Immunol. Methods243:211. Such procedures provide alternatives to traditional hybridomatechniques for the isolation and subsequent cloning of monoclonalantibodies.

Yet other embodiments of the present invention comprise the generationof human or substantially human antibodies in nonhuman animals, such astransgenic animals harboring one or more human immunoglobulintransgenes. Such animals may be used as a source for splenocytes forproducing hybridomas, as is described in U.S. Pat. No. 5,569,825,WO00076310, WO00058499 and WO00037504 and incorporated by referenceherein.

Yet another highly efficient means for generating recombinant antibodiesis disclosed by Newman, Biotechnology, 10: 1455-1460 (1992).Specifically, this technique results in the generation of primatizedantibodies that contain monkey variable domains and human constantsequences. This reference is incorporated by reference in its entiretyherein. Moreover, this technique is also described in commonly assignedU.S. Pat. Nos. 5,658,570, 5,693,780 and 5,756,096 each of which isincorporated herein by reference.

In another embodiment, lymphocytes can be selected by micromanipulationand the variable genes isolated. For example, peripheral bloodmononuclear cells can be isolated from an immunized mammal and culturedfor about 7 days in vitro. The cultures can be screened for specificIgGs that meet the screening criteria. Cells from positive wells can beisolated. Individual Ig-producing B cells can be isolated by FACS or byidentifying them in a complement-mediated hemolytic plaque assay.Ig-producing B cells can be micromanipulated into a tube and the Vh andVl genes can be amplified using, e.g., RT-PCR. The VH and VL genes canbe cloned into an antibody expression vector and transfected into cells(e.g., eukaryotic or prokaryotic cells) for expression.

Alternatively, antibody-producing cell lines may be selected andcultured using techniques well known to the skilled artisan. Suchtechniques are described in a variety of laboratory manuals and primarypublications. In this respect, techniques suitable for use in theinvention as described below are described in Current Protocols inImmunology, Coligan et al., Eds., Green Publishing Associates andWiley-Interscience, John Wiley and Sons, New York (1991) which is hereinincorporated by reference in its entirety, including supplements.

Variable and constant region domains can be obtained from existingsources, (e.g., from one or more of the anti-GITR antibodies describedherein) and be incorporated into a modified binding molecule of theinvention. For example, to clone antibodies, mRNA can be isolated fromhybridoma, spleen, or lymph cells, reverse transcribed into DNA, andantibody genes amplified by PCR. PCR may be initiated by consensusconstant region primers or by more specific primers based on thepublished heavy and light chain DNA and amino acid sequences. PCR alsomay be used to isolate DNA clones encoding the antibody light and heavychains. In this case the libraries may be screened by consensus primersor larger homologous probes, such as mouse constant region probes.Numerous primer sets suitable for amplification of antibody genes areknown in the art (e.g., 5′ primers based on the N-terminal sequence ofpurified antibodies (Benhar and Pastan. 1994. Protein Engineering7:1509); rapid amplification of cDNA ends (Ruberti, F. et al. 1994. J.Immunol. Methods 173:33); antibody leader sequences (Larrick et al. 1989Biochem. Biophys. Res. Commun. 160:1250); or based on known variableregion framework amino acid sequences from the Kabat (Kabat et al. 1991.Sequences of Proteins of Immunological Interest. Bethesda, Md.:JS Dep.Health Hum. Serv. 5^(th) ed.) or the V-base databases (e.g., Orlandi etal. 1989. Proc. Natl. Acad. Sci. USA 86:3833; Sblattero et al. 1998.Immunotechnology 3:271; or Krebber et al. 1997. J. Immunol. Methods201:35). Constant region domains can be selected having a particulareffector function (or lacking a particular effector function) or with aparticular modification to reduce immunogenicity. Variable and constantdomains can be cloned, e.g., using the polymerase chain reaction andprimers which are selected to amplify the domain of interest. PCRamplification methods are described in detail in. U.S. Pat. Nos.4,683,195; 4,683,202; 4,800,159; 4,965,188; and in, e.g., “PCRProtocols: A Guide to Methods and Applications” Innis et al. eds.,Academic Press, San Diego, CA (1990); Ho et al. 1989. Gene 77:51; Hortonet al. 1993. Methods Enzymol. 217:270).

Alternatively, V domains can be obtained from libraries of V genesequences from an animal of choice. Libraries expressing randomcombinations of domains, e.g., VH and VL domains, can be screened with adesired antigen to identify elements which have desired bindingcharacteristics. Methods of such screening are well known in the art.For example, antibody gene repertoires can be cloned into a A,bacteriophage expression vector (Huse, W D et al. 1989. Science2476:1275). In addition, cells (Boder and Wittrup. 1997. Nat.Biotechnol. 15:553; Daugtherty, P. et al. 2000. J. Immunol. Methods.243:211; Francisco et al. 1994. Proc. Natl. Acad. Sci. USA 90:10444;Georgiou et al. 1997. Nature Biotechnology 15:29) or viruses (e.g.,Hoogenboom, H R. 1998 Immunotechnology 4:1 Winter et al. 1994. Annu.Rev. Immunol. 12:433; Griffiths, A D. 1998. Curr. Opin. Biotechnol.9:102) expressing antibodies on their surface can be screened. Ribosomaldisplay can also be used to screen antibody libraries (Hanes J., et al.1998. Proc. Natl. Acad. Sci. USA 95:14130; Hanes, J. and Pluckthun.1999. Curr. Top. Microbiol. Immunol. 243:107; He, M. and Taussig. 1997.Nucleic Acids Research 25:5132).

Preferred libraries for screening are human V gene libraries. VL and VHdomains from a non-human source may also be used. In one embodiment,such non-human V domains can be altered to reduce their immunogenicityusing art recognized techniques.

Libraries can be naïve, from immunized subjects, or semi-synthetic(Hoogenboom, H. R. and Winter. 1992. J. Mol. Biol. 227:381; Griffiths, AD, et al. EMBO J. 13:3245; de Kruif, J. et al. 1995. J. Mol. Biol.248:97; Barbas, C. F., et al. 1992. Proc. Natl. Acad. Sci. USA 89:4457).

In addition, the sequences of many antibody V and C domains are knownand such domains can be synthesized using methods well known in the art.In one embodiment, mutations can be made to immunoglobulin domains tocreate a library of nucleic acid molecules having greater heterogeneity(Thompson, J., et al. 1996. J. Mol. Biol. 256:77; Lamminmaki, U. et al.1999. J. Mol. Biol. 291:589; Caldwell, R. C. and Joyce G F. 1992. PCRMethods Appl. 2:28; Caldwell R C and Joyce G F. 1994. PCR Methods Appl.3:S136. Standard screening procedures can be used to select highaffinity variants. In another embodiment, changes to VH and VL sequencescan be made to increase or decrease antibody avidity, e.g., usinginformation obtained from crystal structures using techniques known inthe art.

Antigen recognition sites or entire variable regions may be derived fromone or more parental antibodies. The parental antibodies can includenaturally occurring antibodies or antibody fragments, antibodies orantibody fragments adapted from naturally occurring antibodies,antibodies constructed de novo using sequences of antibodies or antibodyfragments known to be specific for GITR. Sequences that may be derivedfrom parental antibodies include heavy and/or light chain variableregions and/or CDRs, framework regions or other portions thereof.

In one embodiment, the GITR binding molecule is a humanized antibody. Tomake humanized antibodies, animals are immunized with the desiredantigen, the corresponding antibodies are isolated, and the portion ofthe variable region sequences responsible for specific antigen bindingis removed. The animal-derived antigen binding regions are then clonedinto the appropriate position of human antibody genes in which theantigen binding regions have been deleted. See, e.g. Jones, P. et al.(1986), Nature 321, 522-525 or Tempest et al. (1991) Biotechnology 9,266-273. Also, transgenic mice, or other mammals, may be used to expresshumanized antibodies. Such humanization may be partial or complete.Humanized antibodies minimize the use of heterologous (inter-species)sequences in human antibodies, and are less likely to elicit immuneresponses in the treated subject.

In one embodiment, a binding molecule of the invention comprises orconsists of an antigen binding fragment of an antibody. The term“antigen-binding fragment” refers to a polypeptide fragment of animmunoglobulin or antibody that binds antigen or competes with intactantibody (i.e., with the intact antibody from which they were derived)for antigen binding (i.e., specific binding). As used herein, the term“fragment” of an antibody molecule includes antigen-binding fragments ofantibodies, for example, an antibody light chain (VL), an antibody heavychain (VH), a single chain antibody (scFv), a F(ab′)2 fragment, a Fabfragment, an Fd fragment, an Fv fragment, and a single domain antibodyfragment (DAb). Fragments can be obtained, e.g., via chemical orenzymatic treatment of an intact or complete antibody or antibody chainor by recombinant means.

In one embodiment, a binding molecule of the invention is an engineeredor modified antibody. Engineered forms of antibodies include, forexample, minibodies, diabodies, diabodies fused to CH3 molecules,tetravalent antibodies, intradiabodies (e.g., Jendreyko et al. 2003. J.Biol. Chem. 278:47813), bispecific antibodies, fusion proteins (e.g.,antibody cytokine fusion proteins) or, bispecific antibodies. Otherimmunoglobulins (Ig) and certain variants thereof are described, forexample in U.S. Pat. No. 4,745,055; EP 256,654; Faulkner et al., Nature298:286 (1982); EP 120,694; EP 125,023; Morrison, J. Immun. 123:793(1979); Kohler et al., Proc. Natl. Acad. Sci. USA 77:2197 (1980); Rasoet al., Cancer Res. 41:2073 (1981); Morrison et al., Ann. Rev. Immunol.2:239 (1984); Morrison, Science 229:1202 (1985); Morrison et al., Proc.Natl. Acad. Sci. USA 81:6851 (1984); EP 255,694; EP 266,663; and WO88/03559. Reassorted immunoglobulin chains also are known. See, forexample, U.S. Pat. No. 4,444,878; WO 88/03565; and EP 68,763 andreferences cited therein.

In one embodiment, the modified antibodies of the invention areminibodies. Minibodies are dimeric molecules made up of two polypeptidechains each comprising an ScFv molecule (a single polypeptide comprisingone or more antigen binding sites, e.g., a VL domain linked by aflexible linker to a VH domain fused to a CH3 domain via a connectingpeptide.

ScFv molecules can be constructed in a VH-linker-VL orientation orVL-linker-VH orientation.

The flexible hinge that links the VL and VH domains that make up theantigen binding site preferably comprises from about 10 to about 50amino acid residues. An exemplary connecting peptide for this purpose is(Gly4Ser)3 (Huston et al. 1988. Proc. Natl. Acad. Sci. USA 85:5879).Other connecting peptides are known in the art.

Methods of making single chain antibodies are well known in the art,e.g., Ho et al. 1989. Gene 77:51; Bird et al. 1988 Science 242:423;Pantoliano et al. 1991. Biochemistry 30:10117; Milenic et al. 1991.Cancer Research 51:6363; Takkinen et al. 1991. Protein Engineering4:837.

Minibodies can be made by constructing an ScFv component and connectingpeptide-CH3 component using methods described in the art (see, e.g.,U.S. Pat. No. 5,837,821 or WO 94/09817A1). These components can beisolated from separate plasmids as restriction fragments and thenligated and recloned into an appropriate vector. Appropriate assemblycan be verified by restriction digestion and DNA sequence analysis.

Diabodies are similar to scFv molecules, but usually have a short (lessthan 10 and preferably 1-5) amino acid residue linker connecting bothV-domains, such that the VL and VH domains on the same polypeptide chaincan not interact. Instead, the VL and VH domain of one polypeptide chaininteract with the VH and VL domain (respectively) on a secondpolypeptide chain (WO 02/02781). In one embodiment, a binding moleculeof the invention is a diabody fused to at least one heavy chain portion.In a preferred embodiment, a binding molecule of the invention is adiabody fused to a CH3 domain.

Other forms of modified antibodies are also within the scope of theinstant invention (e.g., WO 02/02781 A1; U.S. Pat. Nos. 5,959,083;6,476,198 B1; US 2002/0103345 A1; WO 00/06605; Byrn et al. 1990. Nature.344:667-70; Chamow and Ashkenazi. 1996. Trends Biotechnol: 14:52).

In one embodiment, a GITR binding molecule of the invention is modifiedto alter one or more glycosylation sites or modified by one or moreother amino acid substitutions that do not alter one or moreglycosylation sites. For example, because the amino acid sequenceAsn-X-(Ser/Thr) is a putative consensus sequence for a glycosylationsite which may affect the production of the binding molecule, aconservative substitution of a glutamine (Gln) for an asparagine (Asn)may be made.

In one embodiment, a binding molecule of the invention comprises animmunoglobulin constant region. It is known in the art that the constantregion mediates several effector functions. For example, binding of theC1 component of complement to binding molecules activates the complementsystem. Activation of complement is important in the opsonisation andlysis of cell pathogens. The activation of complement also stimulatesthe inflammatory response and may also be involved in autoimmunehypersensitivity. Further, binding molecules bind to cells via the Fcregion, with a Fc receptor site on the binding molecule Fc regionbinding to a Fc receptor (FcR) on a cell. There are a number of Fcreceptors which are specific for different classes of binding molecule,including IgG (gamma receptors), IgE (epsilon receptors), IgA (alphareceptors) and IgM (mu receptors). Binding of binding molecule to Fcreceptors on cell surfaces triggers a number of important and diversebiological responses including engulfment and destruction of bindingmolecule-coated particles, clearance of immune complexes, lysis ofbinding molecule-coated target cells by killer cells (calledantibody-dependent cell-mediated cytotoxicity, or ADCC), release ofinflammatory mediators, placental transfer and control of immunoglobulinproduction.

In one embodiment, effector functions may be eliminated or reduced by,for example, using a constant region of an IgG4 binding molecule, whichis thought to be unable to deplete target cells, or making Fc variants,wherein residues in the Fc region critical for effector function(s) aremutated using techniques known in the art, for example, U.S. Pat. No.5,585,097. For example, the deletion or inactivation (through pointmutations or other means) of a constant region domain may reduce Fcreceptor binding of the circulating modified binding molecule therebyincreasing tumor localization. Additionally, amino acid substitutions toremove potential glycosylation sites on Fc may reduce Fc receptorbinding (see, e.g., Shields, et al. (2001) J Biol Chem 276:6591). In oneembodiment, an N297A substitution is made. In another embodiment, aL235A substitution and a L237A is made. In yet another embodiment, aL234A substitution and a L235A substitution is made. In anotherembodiment, a E233P substitution is made. In another embodiment, a L234Vsubstitution is made. In another embodiment, a L235A substitution ismade. In another embodiment, C236 is deleted. In another embodiment, aP238A substitution is made. In another embodiment, a D265A substitutionis made. In another embodiment, a N297A substitution is made. In anotherembodiment, a A327Q substitution is made. In another embodiment, a P329Asubstitution is made. The above recited amino acid positions are basedon the EU numbering system (see, e.g., Kabat, et al. (1991) Sequence ofProteins of Immunological Interest, 5^(th) edition, United States PublicHealth Service, National Institutes of Health, Bethesda).

In other cases it may be that constant region modifications consistentwith the instant invention moderate complement binding and/or reduce theserum half life. Yet other modifications of the constant region may beused to modify disulfide linkages or oligosaccharide moieties that allowfor enhanced localization due to increased antigen specificity orbinding molecule flexibility. More generally, those skilled in the artwill realize that binding molecules modified as described herein mayexert a number of subtle effects that may or may not be readilyappreciated. However the resulting physiological profile,bioavailability and other biochemical effects of the modifications, suchas tumor localization, biodistribution and serum half-life, may easilybe measured and quantified using well know immunological techniqueswithout undue experimentation.

In one embodiment, a binding molecule of the invention can bederivatized or linked to another functional molecule (e.g., anotherpeptide or protein). Accordingly, a binding molecule of the inventioninclude derivatized and otherwise modified forms of the GITR bindingmolecules described herein, including immunoadhesion molecules. Forexample, a binding molecule of the invention can be functionally linked(by chemical coupling, genetic fusion, noncovalent association orotherwise) to one or more other molecular entities, such as anotherbinding molecule (e.g., an scFv antibody, a bispecific antibody or adiabody), a detectable agent, a chemotherapeutic agent (e.g., asdescribed herein), a pharmaceutical agent, and/or a protein or peptidethat can mediate association of the binding molecule with anothermolecule (such as a streptavidin core region or a polyhistidine tag).

In one embodiment, a binding molecule of the invention is modified withpolyethylene glycol. “PEGylation” increases residence time and reducesimmunogenicity in vivo. For example, Knauf et al., J. Biol. Chem., 263:15064 15070 (1988) reported a study of the pharmacodynamic behavior inrats of various polyoxylated glycerol and polyethylene glycol modifiedspecies of interleukin-2. Delgado et al., Br. J. Cancer, 73: 175 182(1996), Kitamura et al., Cancer Res., 51: 4310 4315 (1991), Kitamura etal., Biochem. Biophys. Res. Comm., 171: 1387 1394 (1990), and Pedley etal., Br. J. Cancer, 70: 1126 1130 (1994) reported studies characterizingblood clearance and tissue uptake of certain anti-tumor antigenantibodies or antibody fragments derivatized with low molecular weight(5 kD) PEG. Zapata et al., FASEB J. 9: A1479 (1995) reported that lowmolecular weight (5 or 10 kD) PEG attached to a sulfhydryl group in thehinge region of a Fab′ fragment reduced clearance compared to theparental Fab′ molecule.

One type of derivatized binding molecule is produced by crosslinking twoor more binding molecules (of the same type or of different types, e.g.,to create bispecific antibodies). Suitable crosslinkers include thosethat are heterobifunctional, having two distinctly reactive groupsseparated by an appropriate spacer (e.g.,m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional(e.g., disuccinimidyl suberate). Such linkers are available from PierceChemical Company, Rockford, Ill.

A binding molecule of the invention can be prepared by recombinantexpression of immunoglobulin light and heavy chain genes in a host cell.To express a binding molecule recombinantly, a host cell is transfectedwith one or more recombinant expression vectors carrying DNA fragmentsencoding the immunoglobulin light and heavy chains of the bindingmolecule such that the light and heavy chains are expressed in the hostcell and, preferably, secreted into the medium in which the host cellsare cultured, from which medium a binding molecule can be recovered.Standard recombinant DNA methodologies are used to obtain antibody heavyand light chain genes, incorporate these genes into recombinantexpression vectors, and introduce the vectors into host cells, such asthose described in Sambrook, Fritsch and Maniatis (eds), MolecularCloning; A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y.,(1989), Ausubel, F. M. et al. (eds.) Current Protocols in MolecularBiology, Greene Publishing Associates, (1989) and in U.S. Pat. No.4,816,397 by Boss, et al.

To express a binding molecule of the invention, DNAs encoding partial orfull-length light and heavy chains may be inserted into expressionvector(s) such that the genes are operatively linked to transcriptionaland translational control sequences using methods well known in the art.In this context, the term “operatively linked” means that a bindingmolecule gene is ligated into a vector such that transcriptional andtranslational control sequences within the vector serve their intendedfunction of regulating the transcription and translation of the bindingmolecule gene. In one embodiment, the expression vector and expressioncontrol sequences are chosen to be compatible with the expression hostcell used. The binding molecule light chain gene and the bindingmolecule heavy chain gene may be inserted into separate vector or, moretypically, both genes are inserted into the same expression vector. Thebinding molecule genes may be inserted into the expression vector bystandard methods (e.g., ligation of complementary restriction sites onthe binding molecule gene fragment and vector, or blunt end ligation ifno restriction sites are present). Prior to insertion of the bindingmolecule light or heavy chain sequences, the expression vector mayalready carry binding molecule constant region sequences. For example,one approach to converting VH and VL sequences to full-length bindingmolecule genes is to insert them into expression vectors alreadyencoding heavy chain constant and light chain constant regions,respectively, such that the VH segment is operatively linked to the CHsegment(s) within the vector and the VL segment is operatively linked tothe CL segment within the vector. Additionally or alternatively, therecombinant expression vector can encode a signal peptide thatfacilitates secretion of the binding molecule chain from a host cell.The binding molecule chain gene can be cloned into the vector such thatthe signal peptide is linked in-frame to the amino terminus of thebinding molecule chain gene. The signal peptide can be an immunoglobulinsignal peptide or a heterologous signal peptide (i.e., a signal peptidefrom a non-immunoglobulin protein).

In addition to the binding molecule chain genes, the recombinantexpression vectors of the invention carry regulatory sequences thatcontrol the expression of the binding molecule chain genes in a hostcell. The term “regulatory sequence” includes promoters, enhancers andother expression control elements (e.g., polyadenylation signals) thatcontrol the transcription or translation of the binding molecule chaingenes. Such regulatory sequences are described, for example, in Goeddel;Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990). It will be appreciated by those skilled in theart that the design of the expression vector, including the selection ofregulatory sequences may depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Preferred regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., theadenovirus major late promoter (AdMLP) and polyoma. For furtherdescription of viral regulatory elements, and sequences thereof, seee.g., U.S. Pat. No. 5,168,062 by Stinski, U.S. Pat. No. 4,510,245 byBell et al. and U.S. Pat. No. 4,968,615 by Schaffner, et al.

In addition to the binding molecule chain genes and regulatorysequences, the recombinant expression vectors of the invention may carryadditional sequences, such as sequences that regulate replication of thevector in host cells (e.g., origins of replication) and selectablemarker genes. The selectable marker gene facilitates selection of hostcells into which the vector has been introduced (see e.g., U.S. Pat.Nos. 4,399,216, 4,634,665 and 5,179,017, all by Axel et al.). Forexample, typically the selectable marker gene confers resistance todrugs, such as G418, hygromycin or methotrexate, on a host cell intowhich the vector has been introduced. Preferred selectable marker genesinclude the dihydrofolate reductase (DHFR) gene (for use in dhfr⁻ hostcells with methotrexate selection/amplification) and the neo gene (forG418 selection).

For expression of the light and heavy chains, the expression vector(s)encoding the binding molecule heavy and light chains is transfected intoa host cell by standard techniques. The various forms of the term“transfection” are intended to encompass a wide variety of techniquescommonly used for the introduction of exogenous DNA into a prokaryoticor eukaryotic host cell, e.g., electroporation, calcium-phosphateprecipitation, DEAE-dextran transfection and the like. It is possible toexpress a binding molecule of the invention in either prokaryotic oreukaryotic host cells, expression of binding molecules in eukaryoticcells, and most preferably mammalian host cells, is the most preferredbecause such eukaryotic cells, and in particular mammalian cells, aremore likely than prokaryotic cells to assemble and secrete a properlyfolded and immunologically active binding molecule.

Commonly, expression vectors contain selection markers (e.g.,ampicillin-resistance, hygromycin-resistance, tetracycline resistance orneomycin resistance) to permit detection of those cells transformed withthe desired DNA sequences (see, e.g., Itakura et al., U.S. Pat. No.4,704,362).

E. coli is one prokaryotic host particularly useful for cloning thepolynucleotides (e.g., DNA sequences) of the present invention. Othermicrobial hosts suitable for use include bacilli, such as Bacillussubtilus, and other enterobacteriaceae, such as Salmonella, Serratia,and various Pseudomonas species. In these prokaryotic hosts, one canalso make expression vectors, which will typically contain expressioncontrol sequences compatible with the host cell (e.g., an origin ofreplication). In addition, any number of a variety of well-knownpromoters will be present, such as the lactose promoter system, atryptophan (trp) promoter system, a beta-lactamase promoter system, or apromoter system from phage lambda. The promoters will typically controlexpression, optionally with an operator sequence, and have ribosomebinding site sequences and the like, for initiating and completingtranscription and translation.

Other microbes, such as yeast, are also useful for expression.Saccharomyces is a preferred yeast host, with suitable vectors havingexpression control sequences (e.g., promoters), an origin ofreplication, termination sequences and the like as desired. Typicalpromoters include 3-phosphoglycerate kinase and other glycolyticenzymes. Inducible yeast promoters include, among others, promoters fromalcohol dehydrogenase, isocytochrome C, and enzymes responsible formaltose and galactose utilization.

In addition to microorganisms, mammalian tissue cell culture may also beused to express and produce the polypeptides of the present invention(e.g., polynucleotides encoding binding molecules). See Winnacker, FromGenes to Clones, VCH Publishers, N.Y., N.Y. (1987). Eukaryotic cells areactually preferred, because a number of suitable host cell lines capableof secreting heterologous proteins (e.g., intact binding molecules) havebeen developed in the art, and include CHO cell lines, various Cos celllines, HeLa cells, myeloma cell lines, or transformed B-cells orhybridomas. Preferably, the cells are nonhuman. Expression vectors forthese cells can include expression control sequences, such as an originof replication, a promoter, and an enhancer (Queen et al., Immunol. Rev.89:49 (1986)), and necessary processing information sites, such asribosome binding sites, RNA splice sites, polyadenylation sites, andtranscriptional terminator sequences. Preferred expression controlsequences are promoters derived from immunoglobulin genes, SV40,adenovirus, bovine papilloma virus, cytomegalovirus and the like. See Coet al., J. Immunol. 148:1149 (1992).

Alternatively, binding molecule-coding sequences can be incorporated intransgenes for introduction into the genome of a transgenic animal andsubsequent expression in the milk of the transgenic animal (see, e.g.,Deboer et al., U.S. Pat. No. 5,741,957, Rosen, U.S. Pat. No. 5,304,489,and Meade et al., U.S. Pat. No. 5,849,992). Suitable transgenes includecoding sequences for light and/or heavy chains in operable linkage witha promoter and enhancer from a mammary gland specific gene, such ascasein or beta lactoglobulin.

Preferred mammalian host cells for expressing the recombinant bindingmolecules of the invention include Chinese Hamster Ovary (CHO cells)(including dhfr-CHO cells, described in Urlaub and ChasM, (1980) Proc.Natl. Acad. Sci. USA 77:4216-4220, used with a DHFR selectable marker,e.g., as described in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol.159:601-621), NSO myeloma cells, COS cells and SP2 cells. Whenrecombinant expression vectors encoding binding molecule genes areintroduced into mammalian host cells, binding molecules are produced byculturing the host cells for a period of time sufficient to allow forexpression of the binding molecule in the host cells or, morepreferably, secretion of the binding molecule into the culture medium inwhich the host cells are grown. Binding molecules can be recovered fromthe culture medium using standard protein purification methods.

The vectors containing the polynucleotide sequences of interest (e.g.,the binding molecule heavy and light chain encoding sequences andexpression control sequences) can be transferred into the host cell bywell-known methods, which vary depending on the type of cellular host.For example, calcium chloride transfection is commonly utilized forprokaryotic cells, whereas calcium phosphate treatment, electroporation,lipofection, biolistics or viral-based transfection may be used forother cellular hosts. (See generally Sambrook et al., Molecular Cloning:A Laboratory Manual (Cold Spring Harbor Press, 2nd ed., 1989)(incorporated by reference in its entirety for all purposes). Othermethods used to transform mammalian cells include the use of polybrene,protoplast fusion, liposomes, electroporation, and microinjection (seegenerally, Sambrook et al., supra). For production of transgenicanimals, transgenes can be microinjected into fertilized oocytes, or canbe incorporated into the genome of embryonic stem cells, and the nucleiof such cells transferred into enucleated oocytes.

When heavy and light chains are cloned on separate expression vectors,the vectors are co-transfected to obtain expression and assembly ofintact immunoglobulins. Once expressed, the whole binding molecules,their dimers, individual light and heavy chains, or other immunoglobulinforms of the present invention can be purified according to standardprocedures of the art, including ammonium sulfate precipitation,affinity columns, column chromatography, HPLC purification, gelelectrophoresis and the like (see generally Scopes, Protein Purification(Springer-Verlag, N.Y., (1982)). Substantially pure binding molecules ofat least about 90 to 95% homogeneity are preferred, and 98 to 99% ormore homogeneity most preferred, for pharmaceutical uses.

Host cells can also be used to produce portions of intact bindingmolecules, such as Fab fragments or scFv molecules. It will beunderstood that variations on the above procedure are within the scopeof the present invention. For example, it may be desirable to transfecta host cell with DNA encoding either the light chain or the heavy chain(but not both) of a binding molecule of this invention. Recombinant DNAtechnology may also be used to remove some or all of the DNA encodingeither or both of the light and heavy chains that is not necessary forbinding to GITR. The molecules expressed from such truncated DNAmolecules are also encompassed by a binding molecule of the invention.In addition, bifunctional binding molecules may be produced in which oneheavy and one light chain are a binding molecule of the invention andthe other heavy and light chain are specific for an antigen other thanGITR by crosslinking a binding molecule of the invention to a secondbinding molecule by standard chemical crosslinking methods.

III. Additional Agents

In one embodiment, an additional agent for use in the combinationtherapies of the invention is a chemotherapeutic agent.

Chemotherapeutic agents generally belong to various classes including,for example:

1. Topoisomerase II inhibitors (cytotoxic antibiotics), such as theantracyclines/anthracenediones, e.g., doxorubicin, epirubicin,idarubicin and nemorubicin, the anthraquinones, e.g., mitoxantrone andlosoxantrone, and the podophillotoxines, e.g., etoposide and teniposide;

2. Agents that affect microtubule formation (mitotic inhibitors), suchas plant alkaloids (e.g., a compound belonging to a family of alkaline,nitrogen-containing molecules derived from plants that are biologicallyactive and cytotoxic), e.g., taxanes, e.g., paclitaxel and docetaxel,and the vinka alkaloids, e.g., vinblastine, vincristine, andvinorelbine, and derivatives of podophyllotoxin;

3. Alkylating agents, such as nitrogen mustards, ethyleneiminecompounds, alkyl sulphonates and other compounds with an alkylatingaction such as nitrosoureas, dacarbazine, cyclophosphamide, ifosfamideand melphalan;

4. Antimetabolites (nucleoside inhibitors), for example, folates, e.g.,folic acid, fluropyrimidines, purine or pyrimidine analogues such as5-fluorouracil, capecitabine, gemcitabine, methotrexate and edatrexate;

5. Topoisomerase I inhibitors, such as topotecan, irinotecan, and9-nitrocamptothecin, and camptothecin derivatives; and 6. Platinumcompounds/complexes, such as cisplatin, oxaliplatin, and carbopaltin;

Exemplary chemotherapeutic agents for use in the methods of theinvention include, but are not limited to, amifostine (ethyol),cisplatin, dacarbazine (DTIC), dactinomycin, mechlorethamine (nitrogenmustard), streptozocin, cyclophosphamide, carrnustine (BCNU), lomustine(CCNU), doxorubicin (adriamycin), doxorubicin lipo (doxil), gemcitabine(gemzar), daunorubicin, daunorubicin lipo (daunoxome), procarbazine,mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil (5-FU),vinblastine, vincristine, bleomycin, paclitaxel (taxol), docetaxel(taxotere), aldesleukin, asparaginase, busulfan, carboplatin,cladribine, camptothecin, CPT-11, 10-hydroxy-7-ethyl-camptothecin(SN38), dacarbazine, S-I capecitabine, ftorafur, 5′deoxyflurouridine,UFT, eniluracil, deoxycytidine, 5-azacytosine, 5-azadeoxycytosine,allopurinol, 2-chloro adenosine, trimetrexate, aminopterin,methylene-10-deazaaminopterin (MDAM), oxaplatin, picoplatin,tetraplatin, satraplatin, platinum-DACH, ormaplatin, CI-973, JM-216, andanalogs thereof, epirubicin, etoposide phosphate, 9-aminocamptothecin,10, 11-methylenedioxycamptothecin, karenitecin, 9-nitrocamptothecin, TAS103, vindesine, L-phenylalanine mustard, ifosphamidemefosphamide,perfosfamide, trophosphamide carmustine, semustine, epothilones A-E,tomudex, 6-mercaptopurine, 6-thioguanine, amsacrine, etoposidephosphate, karenitecin, acyclovir, valacyclovir, ganciclovir,amantadine, rimantadine, lamivudine, zidovudine, bevacizumab,trastuzumab, rituximab, 5-Fluorouracil, Capecitabine, Pentostatin,Trimetrexate, Cladribine, floxuridine, fludarabine, hydroxyurea,ifosfamide, idarubicin, mesna, irinotecan, mitoxantrone, topotecan,leuprolide, megestrol, melphalan, mercaptopurine, plicamycin, mitotane,pegaspargase, pentostatin, pipobroman, plicamycin, streptozocin,tamoxifen, teniposide, testolactone, thioguanine, thiotepa, uracilmustard, vinorelbine, chlorambucil, cisplatin, doxorubicin, paclitaxel(taxol) and bleomycin, and combinations thereof which are readilyapparent to one of skill in the art based on the appropriate standard ofcare for a particular tumor or cancer.

In one embodiment, a chemotherapeutic agent for use in the combinationtherapies of the invention is selected from the group consisting ofGemzar, 5-FU, Vincristine, Vinblastine, Adriamycin, Cisplatin, Taxol,Thalidomide, Velcade, methotrexate, cytarabine, fludarabine, hyroxyurea,danorubracin, etopside, mitoxantrone, chlorambucil, cyclophosphamide,melphelan, thiotepa, bleomycin, dacarbazine, L-asparaginase, andprocarbazine.

In one embodiment, a chemotherapeutic agent is a topoisomerase IIinhibitor. In another embodiment, a chemotherapeutic agent is an agentthat affects microtubule formation. In another embodiment, achemotherapeutic agent is an alkylating agent. In another embodiment, achemotherapeutic agent is a topoisomerase I inhibitor. In anotherembodiment, a chemotherapeutic agent is a platinum compound/complex. Inanother embodiment, a chemotherapeutic agent is a hormone, hormonalanalogue, and/or hormonal complex. In another embodiment, achemotherapeutic agent is an enzyme, protein, peptide, polyclonal and/ormonoclonal antibody. In one embodiment, the chemotherapeutic agent foruse in the methods of the invention is an antimetabolite.

The term “antimetabolite” refers to a substance which is structurallysimilar to a critical natural intermediate (metabolite) in a biochemicalpathway leading to DNA or RNA synthesis which is used by the host inthat pathway, but acts to inhibit the completion of that pathway (i.e.,synthesis of DNA or RNA). More specifically, antimetabolites typicallyfunction by (1) competing with metabolites for the catalytic orregulatory site of a key enzyme in DNA or RNA synthesis, or (2)substitute for a metabolite that is normally incorporated into DNA orRNA, and thereby producing a DNA or RNA that cannot support replication.Major categories of antimetabolites include (1) folic acid analogs,which are inhibitors of dihydrofolate reductase (DHFR); (2) purineanalogs, which mimic the natural purines (adenine or guanine) but arestructurally different so they competitively or irreversibly inhibitnuclear processing of DNA or RNA; and (3) pyrimidine analogs, whichmimic the natural pyrimidines (cytosine, thymidine, and uracil), but arestructurally different so thy competitively or irreversibly inhibitnuclear processing of DNA or RNA. Non-limiting examples ofantimetabolites of this invention are 5-Fluorouracil, Floxuradine,Thioguanine, Cytarabine, Fludarabine, 6-Mercaptopurine, Methotrexate,Gemcitabine, Capecitabine, Pentostatin, Trimetrexate, and Cladribine.

In one embodiment, the antimetabolite is the nucleoside analoggemcitabine. In another embodiment, the antimetabolite is the nucleosideanalog fluorouracil.

As used herein, an “agent that affects microtubule formation” or“mitotic inhibitor” is an agent that disrupts microtubulepolymerization. Mitotic inhibitors work by interfering with and haltingmitosis (usually during the M phase of the cell cycle), so that the cellwill no longer divide. In one embodiment, an agent that affectsmicrotubule formation is paclitaxol (Taxol®).

As used herein, an “alkylating agent” is an agent that cross-linksguanine nucleobases in DNA making the strands unable to uncoil andseparate. As this is necessary in DNA replication, the cells can nolonger divide. In one embodiment, an alkylating agent iscyclophosphamide, also known as cytophosphane. Cyclophosphamide is aprodrug.

In another embodiment, an additional agent for use in the combinationtherapies of the invention is a biologic agent.

Biological agents (also called biologics) are the products of abiological system, e.g., an organism, cell, or recombinant system.Examples of such biologic agents include nucleic acid molecules (e.g.,antisense nucleic acid molecules), interferons, interleukins,colony-stimulating factors, antibodies, e.g., monoclonal antibodies,anti-angiogenesis agents, and cytokines. Exemplary biologic agents arediscussed in more detail below and generally belong to various classesincluding, for example

1. Hormones, hormonal analogues, and hormonal complexes, e.g., estrogensand estrogen analogs, progesterone, progesterone analogs and progestins,androgens, adrenocorticosteroids, antiestrogens, antiandrogens,antitestosterones, adrenal steroid inhibitors, and anti-leuteinizinghormones; and

2. Enzymes, proteins, peptides, polyclonal and/or monoclonal antibodies,such as interleukins, interferons, colony stimulating factor, etc.

In one embodiment, the biologic is an interfereon. Interferons (IFN) area type biologic agent that naturally occurs in the body. Interferons arealso produced in the laboratory and given to cancer patients inbiological therapy. They have been shown to improve the way a cancerpatient's immune system acts against cancer cells. Interferons may workdirectly on cancer cells to slow their growth, or they may cause cancercells to change into cells with more normal behavior. Some interferonsmay also stimulate natural killer cells (NK) cells, T cells, andmacrophages—types of white blood cells in the bloodstream that help tofight cancer cells.

In one embodiment, the biologic is an interleukin. Interleukins (IL)stimulate the growth and activity of many immune cells. They areproteins (cytokines and chemokines) that occur naturally in the body,but can also be made in the laboratory. Some interleukins stimulate thegrowth and activity of immune cells, such as lymphocytes, which work todestroy cancer cells.

In another embodiment, the biologic is a colony-stimulating factor.Colony-stimulating factors (CSFs) are proteins given to patients toencourage stem cells within the bone marrow to produce more blood cells.The body constantly needs new. white blood cells, red blood cells, andplatelets, especially when cancer is present. CSFs are given, along withchemotherapy, to help boost the immune system. When cancer patientsreceive chemotherapy, the bone marrow's ability to produce new bloodcells is suppressed, making patients more prone to developinginfections. Parts of the immune system cannot function without bloodcells, thus colony-stimulating factors encourage the bone marrow stemcells to produce white blood cells, platelets, and red blood cells. Withproper cell production, other cancer treatments can continue enablingpatients to safely receive higher doses of chemotherapy.

In another embodiment, the biologic is an antibody. Antibodies, e.g.,monoclonal antibodies, are agents, produced in the laboratory, that bindto cancer cells. When cancer-destroying agents are introduced into thebody, they seek out the antibodies and kill the cancer cells. Monoclonalantibody agents do not destroy healthy cells. Monoclonal antibodiesachieve their therapeutic effect through various mechanisms. They canhave direct effects in producing apoptosis or programmed cell death.They can block growth factor receptors, effectively arrestingproliferation of tumor cells. In cells that express monoclonalantibodies, they can bring about anti-idiotype antibody formation.

Examples of antibodies which may be used in the combination treatment ofthe invention include anti-CD20 antibodies, such as, but not limited to,cetuximab,

Tositumomab, rituximab, and Ibritumomab. Anti-HER2 antibodies may alsobe used in combination with an anti-GITR antibody for the treatment ofcancer. In one embodiment, the anti-HER2 antibody is Trastuzumab(Herceptin). Other examples of antibodies which may be used incombination with an anti-GITR antibody for the treatment of cancerinclude anti-CD52 antibodies (e.g., Alelmtuzumab), anti-CD-22 antibodies(e.g., Epratuzumab), and anti-CD33 antibodies (e.g., Gemtuzumabozogamicin). Anti-VEGF antibodies may also be used in combination withan anti-GITR antibody for the treatment of cancer. In one embodiment,the anti-VEGF antibody is bevacizumab. hi other embodiments, thebiologic agent is an antibody which is an anti-EGFR antibody e.g.,cetuximab. Another example is the anti-glycoprotein 17-1A antibodyedrecolomab.

In another embodiment, the biologic is a cytokine. Cytokine therapy usesproteins (cytokines) to help a subject's immune system recognize anddestroy those cells that are cancerous. Cytokines are produced naturallyin the body by the immune system, but can also be produced in thelaboratory. This therapy is used with advanced melanoma and withadjuvant therapy (therapy given after or in addition to the primarycancer treatment). Cytokine therapy reaches all parts of the body tokill cancer cells and prevent tumors from growing.

In another embodiment, the biologic is a fusion protein. Fusion proteinsmay also be used. For example, recombinant human Apo2L/TRAIL (Genentech)may be used in a combination therapy. Apo2/TRAIL is the first dualpro-apoptotic receptor agonist designed to activate both pro-apoptoticreceptors DR4 and DR5, which are involved in the regulation of apoptosis(programmed cell death).

In one embodiment, the biologic is an antisense nucleic acid molecule.Antisense nucleic acid molecules may also be used in the methods of theinvention. As used herein, an “antisense” nucleic acid comprises anucleotide sequence which is complementary to a “sense” nucleic acidencoding a protein, e.g., complementary to the coding strand of adouble-stranded cDNA molecule, complementary to an mRNA sequence orcomplementary to the coding strand of a gene. Accordingly, an antisensenucleic acid can hydrogen bond to a sense nucleic acid.

In one embodiment, a biologic agent is an siRNA molecule, e.g., of amolecule that enhances angiogenesis, e.g., bFGF, VEGF and EGFR. In oneembodiment, a biologic agent that inhibits angiogenesis mediates RNAi.RNA interference (RNAi) is a post-transcriptional, targetedgene-silencing technique that uses double-stranded RNA (dsRNA) todegrade messenger RNA (mRNA) containing the same sequence as the dsRNA(Sharp, P. A. and Zamore, P. D. 287, 2431-2432 (2000); Zamore, P. D., etal. Cell 101, 25-33 (2000). Tuschl, T. et al. Genes Dev. 13, 3191-3197(1999); Cottrell T R, and Doering T L. 2003. Trends Microbiol. 11:37-43;Bushman F.2003. Mol Therapy. 7:9-10; McManus M T and Sharp P A. 2002.Nat Rev Genet. 3:737-47). The process occurs when an endogenousribonuclease cleaves the longer dsRNA into shorter, e.g., 21- or22-nucleotide-long RNAs, termed small interfering RNAs or siRNAs. Thesmaller RNA segments then mediate the degradation of the target mRNA.Kits for synthesis of RNAi are commercially available from, e.g. NewEngland Biolabs or Ambion. In one embodiment one or more of thechemistries described herein for use in antisense RNA can be employed inmolecules that mediate RNAi.

The use of antisense nucleic acids to downregulate the expression of aparticular protein in a cell is well known in the art (see e.g.,Weintraub, H. et al., Antisense RNA as a molecular tool for geneticanalysis, Reviews-Trends in Genetics, Vol. 1(1) 1986; Askari, F. K. andMcDonnell, W. M. (1996) N. Eng. J. Med. 334:316-318; Bennett, M. R. andSchwartz, S. M. (1995) Circulation 92:1981-1993; Mercola, D. and Cohen,J. S. (1995) Cancer Gene Ther. 2:47-59; Rossi, J. J. (1995) Br. Med.Bull. 51:217-225; Wagner, R. W. (1994) Nature 372:333-335). An antisensenucleic acid molecule comprises a nucleotide sequence that iscomplementary to the coding strand of another nucleic acid molecule(e.g., an mRNA sequence) and accordingly is capable of hydrogen bondingto the coding strand of the other nucleic acid molecule. Antisensesequences complementary to a sequence of an mRNA can be complementary toa sequence found in the coding region of the mRNA, the 5′ or 3′untranslated region of the mRNA or a region bridging the coding regionand an untranslated region (e.g., at the junction of the 5′ untranslatedregion and the coding region). Furthermore, an antisense nucleic acidcan be complementary in sequence to a regulatory region of the geneencoding the mRNA, for instance a transcription initiation sequence orregulatory element. Preferably, an antisense nucleic acid is designed soas to be complementary to a region preceding or spanning the initiationcodon on the coding strand or in the 3′ untranslated region of an mRNA.

Given the coding strand sequences of a molecule that enhancesangiogenesis, antisense nucleic acids of the invention can be designedaccording to the rules of Watson and Crick base pairing. The antisensenucleic acid molecule can be complementary to the entire coding regionof the mRNA, but more preferably is an oligonucleotide which isantisense to only a portion of the coding or noncoding region of themRNA. For example, the antisense oligonucleotide can be complementary tothe region surrounding the translation start site of the mRNA. Anantisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25,30, 35, 40, 45 or 50 nucleotides in length. An antisense nucleic acid ofthe invention can be constructed using chemical synthesis and enzymaticligation reactions using procedures known in the art. For example, anantisense nucleic acid (e.g., an antisense oligonucleotide) can bechemically synthesized using naturally occurring nucleotides orvariously modified nucleotides designed to increase the biologicalstability of the molecules or to increase the physical stability of theduplex formed between the antisense and sense nucleic acids, e.g.,phosphorothioate derivatives and acridine substituted nucleotides can beused. Examples of modified nucleotides which can be used to generate theantisense nucleic acid include 5-fluorouracil, 5-bromouracil,5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxyrnethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine. To inhibit expression in cells, one or moreantisense oligonucleotides can be used. Alternatively, the antisensenucleic acid can be produced biologically using an expression vectorinto which a nucleic acid has been subcloned in an antisense orientation(i.e., RNA transcribed from the inserted nucleic acid will be of anantisense orientation to a target nucleic acid of interest, describedfurther in the following subsection).

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An α-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al. (1987) Nucleic Acids. Res. 15:6625-6641).The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al. (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al. (1987) FEBSLett. 215:327-330).

In another embodiment, an antisense nucleic acid of the invention is acompound that mediates RNAi. RNA interfering agents include, but are notlimited to, nucleic acid molecules including RNA molecules which arehomologous to the target gene or genomic sequence, “short interferingRNA” (siRNA), “short hairpin” or “small hairpin RNA” (snRNA), and smallmolecules which interfere with or inhibit expression of a target gene byRNA interference (RNAi). RNA interference is a post-transcriptional,targeted gene-silencing technique that uses double-stranded RNA (dsRNA)to degrade messenger RNA (mRNA) containing the same sequence as thedsRNA (Sharp, P. A. and Zamore, P. D. 287, 2431-2432 (2000); Zamore, P.D., et al. Cell 101, 25-33 (2000). Tuschl, T. et al. Genes Dev. 13,3191-3197 (1999)). The process occurs when an endogenous ribonucleasecleaves the longer dsRNA into shorter, 21- or 22-nucleotide-long RNAs,termed small interfering RNAs or siRNAs. The smaller RNA segments thenmediate the degradation of the target mRNA. Kits for synthesis of RNAiare commercially available from, e.g. New England Biolabs and Ambion. Inone embodiment one or more of the chemistries described above for use inantisense RNA can be employed.

Nucleic acid molecules encoding molecules that, e.g., inhibitangiogenesis, may be introduced into the subject in a form suitable forexpression of the encoded protein in the cells of the subject may alsobe used in the methods of the invention. Exemplary molecules thatinhibit angiogenesis include, but are not limited to, TSP-1, TSP-2,IFN-α, IFN-γ, angiostatin, endostsin, tumastatin, canstatin, VEGI, PEDF,vasohibin, and the 16 kDa fragment of prolactin 2-Methoxyestradiol (see,Kerbel (2004) J. Clin Invest 114:884, for review).

For example, a full length or partial cDNA sequence is cloned into arecombinant expression vector and the vector is transfected into a cellusing standard molecular biology techniques. The cDNA can be obtained,for example, by amplification using the polymerase chain reaction (PCR)or by screening an appropriate cDNA library. The nucleotide sequences ofthe cDNA can be used for the design of PCR primers that allow foramplification of a cDNA by standard PCR methods or for the design of ahybridization probe that can be used to screen a cDNA library usingstandard hybridization methods. Following isolation or amplification ofthe cDNA, the DNA fragment is introduced into a suitable expressionvector.

Exemplary biologic agents for use in the methods of the inventioninclude, but are not limited to, gefitinib (Iressa), anastrazole,diethylstilbesterol, estradiol, premarin, raloxifene, progesterone,norethynodrel, esthisterone, dimesthisterone, megestrol acetate,medroxyprogesterone acetate, hydroxyprogesterone caproate,norethisterone, methyltestosterone, testosterone, dexamthasone,prednisone, Cortisol, solumedrol, tamoxifen, fulvestrant, toremifene,aminoglutethimide, testolactone, droloxifene, anastrozole, bicalutamide,flutamide, nilutamide, goserelin, flutamide, leuprolide, triptorelin,aminoglutethimide, mitotane, goserelin, cetuximab, erlotinib, imatinib,Tositumomab, Alemtuzumab, Trastuzumab, Gemtuzumab, Rituximab,Ibritumomab tiuxetan, Bevacizumab, Denileukin diftitox, Daclizumab,interferon alpha, interferon beta, anti-4-1BB, anti-4-1BBL, anti-CD40,anti-CD154, anti-OX40, anti-OX40L, anti-CD28, anti-CD80, anti-CD86,anti-CD70, anti-CD27, anti-HVEM, anti-LIGHT, anti-GITRL, anti-CTLA-4,soluble OX40L, soluble 4-1BBL, soluble CD154, soluble GITRL, solubleLIGHT, soluble CD70, soluble CD80, soluble CD86, soluble CTLA4-Ig,GVAX®, and combinations thereof which are readily apparent to one ofskill in the art based on the appropriate standard of care for aparticular tumor or cancer. The soluble forms of agents may be made as,for example fusion proteins, by operatively linking the agent with, forexample, Ig-Fc region.

It should be noted that more than one additional agent, e.g., 1, 2, 3,4, 5, may be administered in combination with a GITR binding molecule.For example, in one embodiment two chemotherapeutic agents may beadministered in combination with a GITR binding molecule. In anotherembodiment, a chemotherapeutic agent, a biologic agent, and a GITRbinding molecule may be administered.

Various forms of the biologic agents may be used. These include, withoutlimitation, such forms as proform molecules, uncharged molecules,molecular complexes, salts, ethers, esters, amides, and the like, whichare biologically activated when implanted, injected or otherwiseinserted into the tumor.

IV. Therapeutic Methods

The present invention further provides methods of administering to thesubject a combination therapy of the invention.

As set forth above, the methods of the present invention, i.e., the useof a GITR binding molecule in combination with a second agent that isuseful in treating cancer, may be used to treat a malignancy or cancerin a subject. Exemplary cancers include: pancreatic cancer, melanoma,breast cancer, lung cancer, bronchial cancer, colorectal cancer,prostate cancer, stomach cancer, ovarian cancer, urinary bladder cancer,brain or central nervous system cancer, peripheral nervous systemcancer, esophageal cancer, cervical cancer, uterine or endometrialcancer, cancer of the oral cavity or pharynx, liver cancer, kidneycancer, testicular cancer, biliary tract cancer, small bowel or appendixcancer, salivary gland cancer, thyroid gland cancer, adrenal glandcancer, osteosarcoma, chondrosarcoma, and cancer of hematologicaltissues.

In one embodiment, the methods of the invention may be used to treatmelanoma. In another embodiment, the methods of the invention may beused to treat solid tumors, e.g., a carcinoma. Examples of solid tumorsthat can be treated by compounds of the present invention, include butare not limited to breast, testicular, lung, ovary, uterine, cervical,pancreatic, non small cell lung (NSCLC), colon, as well as prostate,gastric, skin, stomach, esophagus and bladder cancer. In one embodiment,a solid tumor is an adenocarcinoma, e.g., of the colon. In oneembodiment of the invention, a solid tumor is a colon tumor. In anotherembodiment of the invention, a solid tumor is selected from the groupconsisting of a colon tumor, a lung tumor, a breast tumor, a stomachtumor, a prostate tumor, a cervical tumor, a vaginal tumor, and apancreatic tumor.

In another embodiment, the tumor is selected from the group consistingof Stage I, Stage II, Stage III, and Stage IV tumors. The stage of atumor is readily determined by one of skill in the art using artrecognized methods of staging, such as the size of the tumor, the numberof lymph nodes or other tissues to which the tumor has metastasized,microscopic analyses, histological analyses, etc.

In one embodiment of the invention, the subject combination therapiesare used to treat established tumors, e.g., tumors of sufficient sizesuch that nutrients can no longer permeate to the center of the tumorfrom the subject's vasculature by osmosis and therefore the tumorrequires its own vascular supply to receive nutrients, i.e, avascularized tumor. In another embodiment, the subject combinationtherapies are used to treat, e.g., inhibit the establishment ofsecondary tumor, e.g., metastasis, and/or reduce the size of a tumor,e.g., an established tumor, and/or a secondary tumor, e.g., ametastasis. In yet another embodiment, the subject combination therapiesare used to prevent the establishment of secondary tumors, e.g.,metastasis.

In one embodiment, a combination therapy is used to treat a tumor havingdimensions of at least about 1 mm×1 mm. In another embodiment of theinvention, a combination therapy is used to treat a tumor that is atleast about 0.5 mm×0.5 mm. In other embodiments of the invention thetumor has a volume of at least about 100 mm³. In one embodiment, acombination therapy of the invention is used to treat a tumor that islarge enough to be found by palpation or by imaging techniques wellknown in the art, such as MRI, ultrasound, or CAT scan.

In certain embodiments of the invention, the subject methods result inan inhibition of tumor size more than about 10%, more than about 20%,more than about 30%, more than about 35%, more than about 42%, more thanabout 43%, more than about 44%, more than about 45%, more than about46%, more than about 47%, more than about 48%, more than about 49%, morethan about 50%, more than about 51%, more than about 52%, more thanabout 53%, more than about 54%, more than about 55%, more than about56%, more than about 57%, more than about 58%, more than about 59%, morethan about 60%, more than about 65%, more than about 70%, more thanabout 75%, more than about 80%, more than about 85%, more than about90%, more than about 95%, or more than about 100%. In one embodiment,the administration of a GITR binding molecule, or an antigen-bindingfragment thereof, and at least one chemotherapeutic agent results in a %T/C of about 42% or greater.

In one embodiment, the combination therapies of the invention have asynergistic effect. A “synergistic effect” of two compounds is one inwhich the effect of the combination of the two agents is greater thanthe sum of their individual effects and is statistically different fromthe controls and the single drugs. In another embodiment, thecombination therapies of the invention have an additive effect. An“additive effect” of two compounds is one in which the effect of thecombination of the two agents is the sum of their individual effects andis statistically different from either the controls and/or the singledrugs.

The GITR binding molecule can be administered in a convenient mannersuch as by injection (subcutaneous, intravenous, etc.), oraladministration, inhalation, transdermal application, or rectaladministration. Depending on the route of administration, the activecompound can be coated in a material to protect the compound from theaction of enzymes, acids and other natural conditions which mayinactivate the compound. For example, to administer the agent by otherthan parenteral administration, it may be desirable to coat, orco-administer the agent with, a material to prevent its inactivation.

In general, the at least one additional agent to be administered incombination with the GITR binding molecule will be administered via theroute by which it is routinely administered when used alone. It will beunderstood that the GITR binding molecule and the at least oneadditional agent need not be administered via the same route.

A combination therapy of the present invention can be administered by avariety of methods known in the art, although for many therapeuticapplications, the preferred route/mode of administration is intravenousinjection or infusion. As will be appreciated by the skilled artisan,the route and/or mode of administration will vary depending upon thedesired results. In certain embodiments, the active compound may beprepared with a carrier that will protect the compound against rapidrelease, such as a controlled release formulation, including implants,transdermal patches, and microencapsulated delivery systems.Biodegradable, biocompatible polymers can be used, such as ethylenevinyl acetate, polyanhydrides, polyglycolic acid, collagen,polyorthoesters, and polylactic acid. Many methods for the preparationof such formulations are patented or generally known to those skilled inthe art. See, e.g., Sustained and Controlled Release Drug DeliverySystems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

In certain embodiments, a binding molecule of the invention may beorally administered, for example, with an inert diluent or anassimilable edible carrier. The compound (and other ingredients, ifdesired) may also be enclosed in a hard or soft shell gelatin capsule,compressed into tablets, or incorporated directly into the subject'sdiet. For oral therapeutic administration, the compounds may beincorporated with excipients and used in the form of ingestible tablets,buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers,and the like. To administer a compound of the invention by other thanparenteral administration, it may be necessary to coat the compoundwith, or co-administer the compound with, a material to prevent itsinactivation.

In certain embodiments, the method comprises parenterally administeringan effective amount of a GITR binding molecule and a second agent to asubject. In one embodiment, the method comprises intraarterialadministration of a GITR binding molecule and at least onechemotherapeutic agent to a subject. In other embodiments, the methodcomprises administering an effective amount of a GITR binding moleculeand at least one chemotherapeutic agent directly to the arterial bloodsupply of a tumor in a subject. In one embodiment, the methods compriseadministering an effective amount of a GITR binding molecule and atleast one chemotherapeutic agent directly to the arterial blood supplyof the cancerous tumor using a catheter. In embodiments where a catheteris used to administer a GITR binding molecule and at least onechemotherapeutic agent, the insertion of the catheter may be guided orobserved by fluoroscopy or other method known in the art by whichcatheter insertion may be observed and/or guided. In another embodiment,the method comprises chemoembolization. For example a chemoembolizationmethod may comprise blocking a vessel feeding the cancerous tumor with acomposition comprised of a resin-like material mixed with an oil base(e.g., polyvinyl alcohol in Ethiodol) and one or more chemotherapeuticagents. In still other embodiments, the method comprises systemicadministration of a GITR binding molecule and at least onechemotherapeutic agent to a subject.

In general, chemoembolization or direct intraarterial or intravenousinjection therapy utilizing pharmaceutical compositions of the presentinvention is typically performed in a similar manner, regardless of thesite. Briefly, angiography (a road map of the blood vessels), or morespecifically in certain embodiments, arteriography, of the area to beembolized may be first performed by injecting radiopaque contrastthrough a catheter inserted into an artery or vein (depending on thesite to be embolized or injected) as an X-ray is taken. The catheter maybe inserted either percutaneously or by surgery. The blood vessel may bethen embolized by refluxing pharmaceutical compositions of the presentinvention through the catheter, until flow is observed to cease.Occlusion may be confirmed by repeating the angiogram. In embodimentswhere direct injection is used, the blood vessel is then infused with apharmaceutical composition of the invention in the desired dose.

Embolization therapy generally results in the distribution ofcompositions containing inhibitors throughout the interstices of thetumor or vascular mass to be treated. The physical bulk of the embolicparticles clogging the arterial lumen results in the occlusion of theblood supply. In addition to this effect, the presence of ananti-angiogenic factor(s) prevents the formation of new blood vessels tosupply the tumor or vascular mass, enhancing the devitalizing effect ofcutting off the blood supply. Direct intrarterial, intravenous orinjection administration generally results in distribution ofcompositions containing inhibitors throughout the interstices of thetumor or vascular mass to be treated as well. However, the blood supplyis not generally expected to become occluded with this method.

In one aspect of the present invention, primary and secondary tumors maybe treated utilizing embolization or direct intraarterial or intravenousinjection therapy. Briefly, a catheter is inserted via the femoral orbrachial artery and advanced into the, e.g., hepatic artery, by steeringit through the arterial system under fluoroscopic. guidance. Thecatheter is advanced into the hepatic arterial tree as far as necessaryto allow complete blockage of the blood vessels supplying the tumor(s),while sparing as many of the arterial branches supplying normalstructures as possible. Ideally this will be a segmental branch of thehepatic artery, but it could be that the entire hepatic artery distal tothe origin of the gastroduodenal artery, or even multiple separatearteries, will need to be blocked depending on the extent of tumor andits individual blood supply. Once the desired catheter position isachieved, the artery is embolized by injecting compositions (asdescribed above) through the arterial catheter until flow in the arteryto be blocked ceases, preferably even after observation for 5 minutes.Occlusion of the artery may be confirmed by injecting radio-opaquecontrast through the catheter and demonstrating by fluoroscopy or X-rayfilm that the vessel which previously filled with contrast no longerdoes so. In embodiments where direct injection is used, the artery isinfused by injecting compositions (as described above) through thearterial catheter in a desired dose. The same procedure may be repeatedwith each feeding artery to be occluded.

With respect to dosing, it is to be noted that dosage amounts, number ofcycles administered, and the sequence of administration may vary withthe severity of the condition to be alleviated. It is to be furtherunderstood that for any particular subject, specific dosage regimens maybe adjusted over time according to the individual need and theprofessional judgment of the person administering or supervising theadministration of the compositions. Typically, dosing will be determinedusing techniques known to one skilled in the art. The selected dosagelevel will depend upon a variety of factors including the activity ofthe agent, or the ester, salt or amide thereof, the route ofadministration, the time of administration, the rate of excretion ormetabolism of the particular compound being employed, the duration ofthe treatment, other drugs, compounds and/or materials used incombination with the particular compound employed, the age, sex, weight,condition, general health, disease state, the ability of the bindingmolecule to elicit a desired response in the individual, and priormedical history of the patient being treated, and like factors wellknown in the medical arts.

Dosage regimens may be adjusted to provide the optimum desired response.For example, a single bolus may be administered, several divided dosesmay be administered over time or the dose may be proportionally reducedor increased as indicated by the exigencies of the therapeuticsituation. In one embodiment, one or more of the agents to beadministered may be formulated as a parenteral composition in dosageunit form for ease of administration and uniformity of dosage. Dosageunit form as used herein refers to physically discrete units suited asunitary dosages for the mammalian subjects to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier. The specification for the dosage unit forms ofthe invention are dictated by and directly dependent on (a) the uniquecharacteristics of the active compound and the particular therapeutic orprophylactic effect to be achieved, and (b) the limitations inherent inthe art of compounding such an active compound for the treatment ofsensitivity in individuals.

A physician or veterinarian having ordinary skill in the art can readilydetermine and prescribe the effective amount of each of the agents ofthe subject combination therapy. For example, the physician orveterinarian could administer at least one additional agent at the doseat which it would be administered to the subject if it were to beadministered alone, or as part of a combination therapy that does notemploy a GITR binding molecule. Such art recognized dosing protocols canbe determined by the skilled artisan without undue experimentation.

The combined use of a GITR binding molecule and at least onechemotherapeutic agent as described herein, may reduce the requireddosage for any individual agent. Accordingly, in one embodiment, thedose of at least one additional agent may be lower than that required inorder to achieve the desired therapeutic effect were the agent to beadministered alone.

With respect to GITR binding molecules, one of ordinary skill in the artwould also readily be able to determine an optimal dose. For example, ananti-GITR antibody could be administered at a dose of between about 50mg/kg and about 0.05 mg/kg. In one embodiment, an anti-GITR antibodycould be administered at a dose of between about 40 mg/kg and about 0.1mg/kg. In another embodiment, an anti-GITR antibody could beadministered at a dose of between about 30 mg/kg and about 0.5 mg/kg. Instill another embodiment, an anti-GITR antibody could be administered ata dose of between about 20 mg/kg and about 1 mg/kg. Ranges intermediateto the above recited values are also intended to be part of thisinvention. In yet another embodiment, an anti-GITR antibody could beadministered at a dose of between about 10mg/kg and about 5 mg/kg. Forexample, exemplary doses include: about 0.06, about 0.07, about 0.08,about 0.09, about 0.1, about 0.2, about 0.3, about 0.4, about 0.5, about0.6, about 0.7, about 0.8, about 0.9, about 1, about 1.5, about 2, about2.5, about 3, about 3.5, about 4, about 4.5, about 5, about 10, about20, about 30, or about 40 mg/kg. It is noted that the dosages and dosageranges set forth herein are exemplary only and are not intended to limitthe scope or practice of the claimed composition.

Data obtained from cell culture assays and animal studies may be used informulating a range of dosage for use in humans by for example,determining the dose at which no adverse effects occur in, for example,a mouse, and determining the human equivalent dosage (see, e.g.,www.fda.gov/cber/gdlns/dose.htm, the contents of which are incorporatedherein by reference). The dosage of any supplement, or alternatively ofany components therein, lies preferably within a range of circulatingconcentrations that include the ED50 (median effective dose) with littleor no toxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. Foragents of the present invention, the therapeutically effective dose maybe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC50 (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information may be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

Alternatively, the dosage of the subject invention may be determined byreference to the plasma concentrations of the composition. For example,the maximum plasma concentration (Cmax) and the area under the plasmaconcentration-time curve from time 0 to infinity (AUC (0-4)) may beused. Dosages for the present invention include those that produce theabove values for Cmax and AUC (0-4) and other dosages resulting inlarger or smaller values for those parameters.

The precise time of administration and amount of any particular compoundthat will yield the most effective treatment in a given patient willdepend upon the activity, pharmacokinetics, and bioavailability of aparticular compound, physiological condition of the patient (includingage, sex, disease type and stage, general physical condition,responsiveness to a given dosage and type of medication), route ofadministration, and the like. The guidelines presented herein may beused to optimize the treatment, e.g., determining the optimum timeand/or amount of administration, which will require no more than routineexperimentation consisting of monitoring the subject and adjusting thedosage and/or timing.

In one embodiment, the at least one non-GITR binding agent of thecombination therapy is administered to a subject prior to administrationof the GITR binding molecule. The non-GITR binding molecule may beadministered once or more than once to the patient. In cases of repeatadministration, the non-GITR binding molecule may be administered daily,on alternative days, weekly, monthly, or according to another schedule.An exemplary treatment entails administration in multiple dosages over aprolonged period, for example, of at least six months.

Similarly, a GITR binding molecule of the invention may be administeredonce or more than once to a subject. In cases of repeat administration,the GITR binding molecule may be administered daily, on alternativedays, weekly, monthly, or according to another schedule. An exemplarytreatment entails administration in multiple dosages over a prolongedperiod, for example, of at least six months.

In instances when the non-GITR binding molecule is administered prior toor after the administration of the GITR binding molecule, intervalsbetween administration of the agents can be, e.g., minutes, hours, days,weeks, or months.

A combination therapy of the invention comprising a GITR bindingmolecule and at least one additional agent may optionally includeadministration of additional agents or treatment regimes, e.g., surgery,radiation therapy, that are effective in treating cancer. Preferredadditional agents are those which are art recognized and are routinelyadministered to treat a particular disorder.

While the subject is being treated, the health of the patient may bemonitored by measuring one or more of the relevant indices atpredetermined times, e.g., during a 24-hour period. Treatment, includingsupplement, amounts, times of administration and formulation, may beoptimized according to the results of such monitoring. The patient maybe periodically reevaluated to determine the extent of improvement bymeasuring the same parameters, the first such reevaluation typicallyoccurring at the end of four weeks from the onset of therapy, andsubsequent reevaluations occurring every four to eight weeks duringtherapy and then every three months thereafter. Therapy may continue forseveral months or even years, with a minimum of one month being atypical length of therapy for humans. Adjustments to the amount(s) ofagent administered and possibly to the time of administration may bemade based on these reevaluations.

In one embodiment, the GITR binding molecule and the second agent areconjugated using methods known in the art.

V. Kits of the Invention

The present invention provides kits and articles of manufacture for useof the methods of the present invention. The invention also pertains topackaged pharmaceutical compositions or kits for administering the GITRbinding molecule and a second agent used in the invention for thetreatment of cancer. In one embodiment of the invention, the kit orarticle of manufacture, comprises a GITR binding molecule, andinstructions for administration for treatment of cancer in combinationwith at least one additional agent, e.g., a chemotherapeutic agent. Inanother embodiment, the kit comprises a second container comprising atleast one additional agent for use in a combination therapy with theGITR binding molecule. The instructions may describe how, e.g.,intravenously, and when, e.g., at week 0 and week 2, the different dosesof GITR binding molecule and at least one chemotherapeutic agent shallbe administered to a subject for treatment.

The package or kit alternatively can contain the GITR binding moleculeand it can be promoted for use, either within the package or throughaccompanying information, for the uses or treatment of the disordersdescribed herein. The packaged pharmaceuticals or kits further caninclude a chemotherapeutic agent (as described herein) packaged with orco-promoted with instructions for using the second agent, e.g., achemotherapeutic agent, with a first agent, e.g. a GITR bindingmolecule.

For example, a kit may comprise a packaging material, one or more GITRbinding molecules and at least one chemotherapeutic agent as describedabove and optionally a label or package insert. In still otherembodiments, the invention provides a kit comprising one or more GITRbinding molecules and at least one chemotherapeutic agent and one ormore devices for accomplishing administration of such compositions. Forexample, a kit may comprise a pharmaceutical composition comprising aGITR binding molecule and catheter for accomplishing directintraarterial injection of the composition into a solid tumor. The kitsoptionally include accessory components such as a second containercomprising a pharmaceutically-acceptable buffer and instructions forusing the composition.

This invention is further illustrated by the following examples, whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication, as well as the Figures, are incorporated herein byreference.

EXAMPLES Example 1 The Combination of a GITR Binding Molecule and aNucleoside Analog Decreases Tumor Burden and Increases Survival Time inan Animal Model of Colon Carcinoma.

Mice were injected in the flank with 1×10⁵ CT26 cells and divided intogroups. One group of control mice was untreated. The group of micereceiving Gemzar was treated with 80 mg/kg Gemzar on Day 15. One groupof mice received anti-GITR antibody (2F8) alone at a dose of 0.4 mg(I.P.) on Days 15, 16 and 17. The group of mice receiving Gemzar+2F8were given 80 mg/kg Gemzar on Day 15 and 0.4 mg of 2F8 (I.P.) on Day 16.

The size of the tumors and the survival of the mice were monitored.GraphPad Prism 4 software was used to plot Kaplan-Meir survival curvesand to confirm the median survival times for the groups. Tumors weremeasured using calipers, and tumor size was calculated using the formula(L×W²)/2.

Tumor burden in mice treated with the combination of Gemzar and 2F8 wasreduced as compared to the tumor burden of mice treated with vehicle,Gemzar alone, or 2F8 alone (FIG. 1).

In addition, as shown in FIG. 2, the median survival for the IgG2acontrol mice and the mice treated with the GITR binding molecule alonewas 24 days. Median survival was 31 days for the mice treated withGemzar alone. All the Gemzar plus 2F8 treated mice were still alive atday 31 and the tumors were moderate in size.

In a second study, mice were injected in the tail vein with 1×10⁵ CT26cells and tumors were allowed to establish in the lung for 10 days. Theanimals were then divided into groups. One group of control mice wasuntreated. The group of mice receiving Gemzar was treated with 80 mg/kgGemzar on Day 15. One group of mice received anti-GITR antibody (2F8)alone at a dose of 0.4 mg (I.P.) on Days 15, 16 and 17. The group ofmice receiving Gemzar+2F8 were given 80 mg/kg Gemzar on Day 15 and 0.4mg of 2F8 (I.P.) on Day 16. The number of tumors was assessed on day 22.

As shown in FIG. 3, the number of tumors in mice treated with thecombination of Gemzar and 2F8 was reduced as compared to the number oftumors in mice treated with vehicle, Gemzar alone, or 2F8 alone.

Example 2 The Combination of a GITR Binding Molecule and an Agent thatAffects Microtubule Formation Decreases Tumor Burden in an Animal Modelof Melanoma

Mice were injected in the flank with 12×10³ B16 melanoma cells anddivided into groups. One group of control mice was untreated. The groupof mice receiving Taxol® was treated with 10 mg/kg Taxol® on Day 20 whentumors were approximately 100 mm³. One group of mice received anti-GITRantibody (2F8) alone at a dose of 0.4 mg (I.P.) on Day 21. The group ofmice receiving Taxol®+2F8 were given 10 mg/kg Taxol® on Day 20 and 0.4mg of 2F8 (I.P.) on Day 21.

The size of the tumors and the survival of the mice were monitored.Tumors were measured using calipers, and tumor size was calculated usingthe formula (L×W²)/2.

As shown in FIG. 4, the tumor burden in mice treated with thecombination of Taxol® and 2F8 was reduced as compared to the tumorburden of mice treated with vehicle, Taxol® alone, or 2F8 alone.

Example 3 The Combination of a GITR Binding Molecule and an AlkylatingAgent Decreases Tumor Burden in an Animal Model of Colon Carcinoma

Mice were injected subcutaneously with 1×10⁵ CT26 cells and divided intogroups. One group of control mice was untreated. The group of micereceiving Cytoxan was treated with 150 mg/kg Cytoxan on Day 13. Onegroup of mice received anti-GITR antibody (2F8) alone at a dose of 0.4mg (I.P.) on Day 14. The group of mice receiving Cytoxan+2F8 were given150 mg/kg Cytoxan® on Day 13 and 0.4 mg of 2F8 (LP.) on Day 14.

The size of the tumors and the survival of the mice were monitored.Tumors were measured using calipers, and tumor size was calculated usingthe formula (L×W²)/2.

As shown in FIG. 5, the tumor burden in mice treated with thecombination of Cytoxan and 2F8 was reduced as compared to the tumorburden of mice treated with vehicle or Cytoxan alone.

Example 4 An Animal Model of Colon Carcinoma Treated with theCombination of a GITR Binding Molecule and an Alkylating Agent or aNucleoside Analog Develop a Robust Memory Response to CT26 Cells

Mice treated as above in Examples 1 and 4 that had complete remission oftheir tumors were used in studies where they were injected with 3×10⁵CT26 cells IV (4 mice) or 10⁶ CT26 cells on their left flank and 10⁶RENCA cells on their right flank (4 mice). Mice naïve to CT26 were usedas controls. All 4 combination treated mice rejected the CT26 cellchallenge and 2/4 completely rejected the RENCA cells. In the IV study,lungs were resected 14 days after injection of cells, stained with Indiaink and fixed with Fekete's Solution and analyzed for the presence oftumors; analysis of the lungs of all 4 animals showed no visible signsof tumors.

Example 5 The Combination of a GITR Binding Molecule and anAntimetabolite Decreases Tumor Burden in an Animal Model of ColonCarcinoma

Mice were injected subcutaneously with 1×10⁵ CT26 cells and divided intogroups. One group of control mice was untreated. The group of micereceiving fluorouracil (5-FU) was treated with 75 mg/kg 5-FU on Day 10.The group of mice receiving 5-FU+2F8 were given 75 mg/kg 5-FU on Day 10and 0.4 mg of 2F8 (I.P.) on Day 11.

The size of the tumors and the survival of the mice were monitored.Tumors were measured using calipers, and tumor size was calculated usingthe formula (L×W²)/2.

As shown in FIG. 6, the tumor burden in mice treated with thecombination of 5-FU and 2F8 was reduced as compared to the tumor burdenof mice treated with vehicle or 5-FU alone.

Example 6 The Combination of a GITR Binding Molecule and a CytotoxicAntibiotic Decreases Tumor Burden in an Animal Model of Colon Carcinoma

Mice were injected subcutaneously with 1×10⁵ CT26 cells and divided intogroups. One group of control mice was untreated. The group of micereceiving doxorubicin (Adriamycin) was treated with 5 mg/kg doxorubicinon Day 10. The group of mice receiving doxorubicin+2F8 were given 5mg/kg doxorubicin on Day 10 and 0.4 mg of 2F8 (I.P.) on Day 11.

The size of the tumors and the survival of the mice were monitored.Tumors were measured using calipers, and tumor size was calculated usingthe formula (L×W²)/2.

As shown in FIG. 7, the tumor burden in mice treated with thecombination of doxorubicin and 2F8 was reduced as compared to the tumorburden of mice treated with vehicle or doxorubicin alone.

Example 7 The Combination of a GITR Binding Molecule and an AlkylatingAgent Decreases Tumor Burden in an Animal Model of Melanoma

Mice were injected subcutaneously with 1×10⁴ B16 mealnoma cells anddivided into groups. One group of control mice was untreated. The groupof mice receiving Cytoxan was treated with 150 mg/kg Cytoxan on Day 13.One group of mice received anti-GITR antibody (2F8) alone at a dose of0.4 mg (I.P.) on Day 14. The group of mice receiving Cytoxan+2F8 weregiven 150 mg/kg Cytoxan® on Day 13 and 0.4 mg of 2F8 (I.P.) on Day 14.

The size of the tumors and the survival of the mice were monitored.Tumors were measured using calipers, and tumor size was calculated usingthe formula (L×W²)/2.

As shown in FIG. 8, the tumor burden in mice treated with thecombination of Cytoxan and 2F8 was reduced as compared to the tumorburden of mice treated with vehicle or Cytoxan alone.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All publications and patents mentioned herein, including those itemslisted below, are hereby incorporated by reference in their entirety asif each individual publication or patent was specifically andindividually indicated to be incorporated by reference.

1.-64. (canceled)
 65. A method for treating a subject having a tumor,the method comprising administering to the subject: a) a GITR-bindingantibody comprising a heavy chain and a light chain, or anantigen-binding fragment thereof, wherein the GITR-binding antibody orthe antigen-binding fragment acts as a GITR agonist; b) achemotherapeutic agent; and c) an anti-CTLA-4 antibody, or anantigen-binding fragment.
 66. The method of claim 65, wherein theGITR-binding antibody or antigen-binding fragment comprises: the heavychain complementarity determining regions (CDRs) set forth in SEQ IDNOs.: 1, 2, and 4 or in SEQ ID NOs.: 1, 3, and 4; and the light chainCDRs set forth in SEQ ID NOs.: 5, 6, and
 7. 67. The method of claim 65,wherein the GITR-binding antibody or antigen-binding fragment compriseshuman heavy chain framework regions.
 68. The method of claim 65, whereinthe GITR-binding antibody or antigen-binding fragment comprises humanlight chain framework regions.
 69. The method of claim 65, wherein theGITR-binding antibody or antigen-binding fragment is humanized.
 70. Themethod of claim 65, wherein the GITR-binding antibody or antigen-bindingfragment is a chimeric antibody or a chimeric antigen-binding fragment.71. The method of claim 65, wherein the GITR-binding antibody orantigen-binding fragment comprises human heavy chain and human lightchain framework regions, except that one or more human framework aminoacid residues is backmutated to a corresponding murine amino acidresidue.
 72. The method of claim 65, wherein the GITR agonist activityof the GITR-binding antibody or the antigen-binding fragment comprisesincreasing T cell effector responses.
 73. The method of claim 65,wherein the chemotherapeutic agent is an antimetabolite, an agent thataffects microtubule formation, an alkylating agent, or a cytotoxicantibiotic.
 74. The method of claim 73, wherein the chemotherapeuticagent is an antimetabolite.
 75. The method of claim 74, wherein theantimetabolite is selected from the group consisting of Aminopterin,Methotrexate, Pemetrexed, Raltitrexed, Cladribine, Clofarabine,Fludarabine, Mercaptopurine, Pentostatin, Thioguanine, Capecitabine,Cytarabine, Fluorouracil, Floxuridine, and Gemcitabine.
 76. The methodof claim 75, wherein the antimetabolite is Gemcitabine.
 77. The methodof claim 65, wherein the GITR-binding antibody or antigen-binding, actssynergistically with the chemotherapeutic agent and the CTLA-4-bindingantibody or antigen-binding fragment.
 78. The method of claim 65,wherein the method results in one or more of the following: inhibitionof tumor growth; reduction in tumor size; reduction in the number oftumors; and decreased tumor burden in the subject.
 79. The method ofclaim 65, wherein the method prolongs survival of the subject.
 80. Amethod for treating a subject having a tumor, the method comprisingadministering to the subject: a) a GITR-binding antibody comprising aheavy chain and a light chain, or GITR-binding fragment thereof, whereinthe GITR-binding antibody or the antigen-binding fragment acts as a GITRagonist, and wherein the GITR-binding antibody or antigen-bindingfragment comprises: the heavy chain complementarity determining regions(CDRs) set forth in SEQ ID NOs.: 1, 2, and 4 or in SEQ ID NOs.: 1, 3,and 4; and the light chain CDRs set forth in SEQ ID NOs.: 5, 6, and 7;b) Gemcitabine; and c) an anti-CTLA-4 antibody, or a CTLA-4-bindingfragment thereof.
 81. The method of claim 80, wherein the GITR-bindingantibody or antigen-binding fragment comprises human framework regions.82. The method of claim 80, wherein the GITR-binding antibody orantigen-binding fragment is humanized.
 83. The method of claim 80,wherein the GITR-binding antibody or antigen-binding fragment is achimeric antibody or a chimeric antigen-binding fragment.
 84. A kit fortreating a subject having a tumor, the kit comprising: a) a GITR-bindingantibody comprising a heavy chain and a light chain, or GITR-bindingfragment thereof, wherein the GITR-binding antibody or theantigen-binding fragment acts as a GITR agonist, and wherein theGITR-binding antibody or antigen-binding fragment comprises: the heavychain complementarity determining regions (CDRs) set forth in SEQ IDNOs.: 1, 2, and 4 or in SEQ ID NOs.: 1, 3, and 4; and the light chainCDRs set forth in SEQ ID NOs.: 5, 6, and 7; b) Gemcitabine; and c) ananti-CTLA-4 antibody, or a CTLA-4-binding fragment thereof.